Students who used the world’s oldest computer during their time at the University of Wolverhampton are being invited on a special visit..
Two West Midlands primary schools are reopening today as academies under a new trust led by the University of Wolverhampton.
More than seven out of ten women who are sleeping rough in the UK say they experienced abuse from their partners, says new research previewed..
The impact of Government cuts on the emergency services will be the focus of a public lecture at the University of Wolverhampton.
A drug that has been used to treat alcoholic patients for more than 60 years could potentially help treat glioblastoma – .
A sports psychologist from the University of Wolverhampton has provided answers to unusual questions about human emotions in a..
The University of Wolverhampton has secured £1.4 million from the Government to enable regional businesses to grow and create new jobs, it was announced today.
Strictly Come Dancing’s Denise Van Outen has suffered a whiplash injury in training and has been described as “touch and go” for Saturday night’s show.
A study by a team of researchers at the University of Wolverhampton has found practitioners with Early Years Professional Status (EYPS) are making a positive..
An innovative project is to be launched by the University of Wolverhampton to help tackle environmental problems in West Africa.
Art and poetry to celebrate inspirational black achievers who have become iconic in their field is to be showcased at the University of Wolverhampton.
A team of experts from an organisation which supports victims of conflicts around the world has completed a specially devised training programme..
The roar of the home crowd at the London 2012 Games is helping to spur athletes on - but also causing some members of Team GB to feel the need to apologise.
A team of education experts from Haiti have visited the University of Wolverhampton to examine ways of working with regional businesses.
The Governor of the Bank of England, Sir Mervyn King, has visited Wolverhampton to present a talk on the economy.
People interested in developing their career by studying part-time at the University of Wolverhampton Business School are invited to an open evening.
A partnership between a Shropshire company and the University of Wolverhampton which helped transform the business has won..
A new online learning tool developed by University of Wolverhampton experts which is aimed at Deaf people ..
The University of Wolverhampton is backing a campaign to urge the Government to do more to support international student recruitment.
Young people with special educational needs and disability from the Black Country are getting set for a talent-spotting sports event..
A new multi million pound University Technical College (UTC) for young people in the West Midlands will open in September 2014..
The volatile global financial market and uncertainty in the Eurozone will be the focus of a special event at the University of Wolverhampton.
The man behind one of the country’s most successful house builders gave staff and students an insight into his business career..
Research into the changing shape of world-class sprinters by University of Wolverhampton experts has been included in a new..
A University of Wolverhampton sports psychologist has helped to develop an online test for BBC Lab UK’s biggest ever experiment.
Over 200 dance professionals joined dance medicine and science leaders at the Royal Society of Medicine..
Dr Amanda Bayley, of the University’s Department of Music, has been carrying out research into the..
Construction students at the University of Wolverhampton are celebrating after clinching second..
A company that has developed environmentally friendly roof tiles has been selected...
Students have been working with Black Country businesses on an innovative pilot scheme to boost entrepreneurial skills.
The University of Wolverhampton is delighted to have been selected as a training camp for the Australian Judo team for this summer’s Olympics.
The University of Wolverhampton Students’ Union is celebrating after being named Most Improved Union at the National Union..
Distinguished figures from the worlds of human rights, law, education and business will take centre stage for..


Chapin 30600 2-Gallon Professional Tri-Poxy Steel Deck Sprayer
The Chapin 30600 2-Gallon Professional Tri-Poxy Steel Deck Sprayer is the perfect tool for any professional deck sprayer. Constructed with a rust-resistant and full tri-poxy steel tank, it can withstand whatever you throw! Its wide mouth opening makes refilling and cleaning effortless, while its ergonomic handle provides superior comfort during pumping and carrying tasks. This heavy-duty solution will make any deck or wood finishing job easy.
Chapin International 1949 Industrial Open Head Sprayer
It is introducing the Chapin International 1949 Industrial Open Head Sprayer, designed to tackle industrial and commercial tasks easily! This robust sprayer features a rust-resistant 2-gallon tri-poxy steel tank that can withstand tough conditions. Additionally, its comfortable handle allows for easy pumping action, while its wide mouth opening provides quick filling and cleaning. And don’t forget the heavy-duty shut-off valve with a lock-on feature for extended use! Get ready to achieve optimal results – this high-performance sprayer is here to stay.
Wagner Spraytech 0518080 Control Spray Max HVLP Paint or Stain Sprayer
Make painting and staining project a breeze with the Wagner Spraytech 0518080 Control Spray Max HVLP Paint or Stain Sprayer! This high-powered, two-stage turbine sprayer has an adjustable air cap for superior atomization and control. Plus, its lightweight design is easy to maneuver around tight spaces. And it comes complete with 20ft of flexible hose, a 1-quart cup, and 3 nozzles offering versatility in spraying patterns. Perfect for DIYers and pros alike – don’t miss out on this incredible tool.
Deck & Home 190399 Universal Sprayer, 2 Gallon
The Deck & Home 190399 Universal Sprayer was engineered to meet the demands of professionals and DIYers. This powerful 2-gallon sprayer is constructed with a polyethylene tank that provides exceptional resistance against harsh chemicals, making it perfect for an array of the deck, home and garden jobs. Moreover, its ergonomic handle ensures simple pumping every time, while its large mouth opening makes filling or cleaning hassle-free. Furthermore, this top-notch tool includes a universal nozzle that can be adjusted to produce different spraying patterns according to your needs.
Graco Magnum 262800 X5 Stand Airless Paint Sprayer, Blue
The Graco Magnum 262800 X5 Stand Airless Paint Sprayer is a heavy-duty, professional-grade painting tool designed for interior and exterior projects. This robust and reliable sprayer comes with a stainless steel piston pump that can effortlessly handle various paints, stains and coatings – making it perfect for large-scale work such as decks, fences or even entire homes. This paint gun comes with an ergonomic handle to provide you with maximum comfort while in use and also features foldable stand-fitted wheels, so it’s easy to transport & store away when not in use.
CHAPIN 20002 2 Gallon Lawn, Sprayer, Translucent White
The CHAPIN 20002 2 Gallon Lawn Sprayer is a powerful, multi-functional sprayer that makes lawn care and gardening effortless. Crafted with premium, chemical-resistant white polyethylene, it’s easy to monitor the liquid level inside its generous 2-gallon tank. It boasts an ergonomic handle for convenient pumping and a wide mouth opening for stress-free filling and washing, all while providing versatility due to its adjustable cone nozzle, which can customize different spraying patterns.
HomeRight C800971.A Super Finish Max HVLP Paint Sprayer
The HomeRight C800971.A Super Finish Max HVLP Paint Sprayer is the perfect tool for DIYers and professionals! With a powerful two-stage turbine that ensures superior atomization and control, this sprayer can easily handle any indoor or outdoor painting job–from furniture to cabinets, walls, and more. Its lightweight design allows you to maneuver it seamlessly, while its ergonomic grip gives you maximum comfort as you work. Plus, its adjustable hose adapts quickly to fit all your project needs! Get ready for some beautiful results with the HomeRight paint sprayer.
VIVOSUN 2-Gallon Pump Pressure Sprayer, Pressurized Lawn & Garden Water Spray Bottle
The VIVOSUN 2-Gallon Pump Pressure Sprayer is the perfect choice for professionals and homeowners alike. Its durable, chemical-resistant polyethylene tank offers a generous 2 gallons capacity to easily tackle any lawn or garden application! Plus, its ergonomic handle makes pumping comfortable and effortless, while its wide mouth opening lets you refill it quickly and keep it clean easily. Not to mention, this sprayer also has an adjustable nozzle,, so you can customize your spraying pattern according to what task at hand.
Smith Performance Sprayers 190447 2 Gallon Bleach Sprayer
The VIVOSUN 2-Gallon Pump Pressure Sprayer is a perfect tool for any professional or home gardener. Its highly durable and chemical-resistant translucent polyethylene tank can handle many applications! The ergonomic handle heightens comfort during use and an adjustable nozzle for various spray patterns. Plus, with its wide mouth opening, you’ll never have to worry about overfilling or cleaning your pump pressure sprayer again. Get ready to revolutionize the way you garden today.
Graco TC Pro Cordless Airless Paint Sprayer
The Smith Performance Sprayers 190447 2 Gallon Bleach Sprayer is a robust sprayer ideally suited for bleach-based cleaning solutions across large surfaces such as patios, decks, driveways and walkways. It features an impressively durable and chemically resistant two-gallon polyethylene tank that can be easily used on numerous projects.


text:
iOS app to show country info and flag
CountryTourList fetch and display country info using Country Layer API.
App will show all the countries along with name, capital and flag image.
You can navigate to the details by tapping on a country.
- Written entirely with UIKit.
- 100% programmatic UI.
- Image Caching.
- Dark/Light mode.
- No third-party libraries.

domain: geography


text:
“Truly wonderful the mind of a child is.” – Yoda
Star Wars is an epic for a reason—the story is long, the heroes (and villains) are legendary, and the movies have captivated fans across generations. Yes, that includes kids. So why not create an epic space for your little Star Wars fan? Whether you’re moving into a new home, furnishing a vacation home, or refurnishing your current home, a Star Wars-themed room is a great way to get your child excited about their space. Here are the four simple steps you’ll want to take to create a space where your child can feel truly at home while they try to make their way in the universe.
Step 1: Planning Your Color Scheme
Light Side, Dark Side, or Both?
If you’re a fan of Star Wars yourself, you probably have a favorite character, team, and possibly even a favorite vehicle. However, children can be less selective. Find out if your Star Wars fan has a favorite character (or several) and take note.
For the child with only one favorite, use that character's design as a guide for your color scheme. For the child with more than one favorite, you may have to do a little research. Whether they love Yoda, Luke, Chewbacca, Boba Fett, Darth Vader, or that one indistinguishable Stormtrooper from that one scene of that one Episode (they'll know the one), try to find the connection between their favorite characters. Are they all on the light side of the Force? The dark side? Is there an Episode they’re all in together? If your at a loss, a quick Google search will provide plenty of inspiration for an ideal color scheme.
The light side is exactly that—light. Preference for characters on the light side of the Force will generally warrant a lighter and brighter color scheme focused on:
- Sandy Light Brown
- Light or Bright Blue
- Lime Green
- Yellow / Gold
You will want to select one of the above as the main color for your child’s Star Wars bedroom and two to four others as accent colors.
Orange, red, and darker colors, such as dark blue and dark brown, should not be used as primary colors for light side-themed rooms unless the design of your child’s favorite character utilizes one of those colors. For example, if your child’s favorite character hands down is BB-8, you’ll want to focus on white, orange, gray, and black.
If your child prefers the dark side, you will want to consider a darker color scheme so their room can mirror and enhance their favorite characters. This will likely include:
- Light or Medium Gray / Silver
- Bright Red
- Royal Purple
Black is the staple of the dark side, which makes it an easy choice to use as the primary color for the room. You might paint the walls light or medium gray and select black furniture to fill the space.
Since this is the dark side, try to use white only as an accent color unless your child’s favorite character is a Stormtrooper. Provided your child’s favorite character is not one of the few on the dark side whose designs use additional colors, stick to the above list for an authentic dark side theme.
Where Light Meets Dark
If your child has no preference, loves the thrill of the battles, or equally favors characters from both sides, use a neutral gray as your base color and select two to four accent colors, evenly from each side, that look good together. This will allow the varying light and dark accents in the room to shine without one side overshadowing the other.
Step 2: Painting
Take a look at the colors you wound up with from Step 1 and think about which color you want to use as the main color for the walls. As mentioned above, you may want to paint the walls light gray for a dark side theme since this is going to be a child’s bedroom. However, dark colors are great if you are planning to add lighter accents over them, such as stars, light beams, or primarily white posters.
Alternatively, you could paint three walls white and the fourth wall dark blue, black, etc, or do the same with different shades of the same color. You could add a mural or have spacecraft flying across the walls as they would through space. To make the room feel bigger and feature two primary colors, consider splitting colors diagonally across two walls that are parallel from each other, so one side of the room is one color and the other is a second color. With an open mind and some skill with a paint brush, the options are endless.
Step 3: Furniture
When furnishing your child’s Star Wars-themed room, it’s okay and often preferable to use traditional furniture instead of Star Wars furniture. Why? Authentic Star Wars furniture is expensive—a single piece can easily run you from nearly $1,000 to upwards of $4,000—and better suited as an accent piece for a more “adult” room.
Remember: You’ll be decorating the room after it’s furnished.
By using traditional furniture for your child’s Star Wars-themed bedroom, you’re creating a blank canvas of essential items that will provide a break from the Star Wars figures, pillows, and other décor. This will help the Star Wars theme stand out without being overwhelming.
If you’re furnishing your entire home at the same time, look through some Florida furniture packages to take the stress out of furnishing a whole house by yourself. Complete home furniture packages like ours allow you to enjoy the experience of furnishing a home while working with professional interior designers who will manage every last detail. Once your home is fully furnished, you can celebrate your beautifully decorated home, happily looking back on how simple the whole process was.
Step 4: Décor
This is the fun part where you get to add all the Star Wars elements to the room. Look for bedding, posters, and an area rug that are in line with your child’s favorite characters. The colors and art styles should match or compliment each other while fitting the color scheme since each item will be adding to the Star Wars story you’re building for your child. For example, if your colors are gray, white, dark blue, and brown, you might paint the walls gray, find brown furniture, look for primarily dark blue Star Wars bedding, and add white, blue, and black décor.
Consider hanging lightsabers or masks on the wall and adding a plush Star Wars character to the bed. A Star Wars quote is another great addition to any wall, whether it’s from a character they like or the opening title sequence. Add solid curtains, a lamp, clock, and other accent items in the accent colors you’ve chosen. If the process feels overwhelming, a skilled interior designer will be able to take the load off your shoulders.
Revealing the Star Wars-Themed Bedroom
You’re done! Take a step back and look at the Star Wars base you’ve created for your child. If everything’s in order, go get them and show them their new Star Wars-themed bedroom. You can expect an excited and grateful child, who is both in awe of the wonder that is now their bedroom and proud to have such a cool space to call their own.

domain: entertainment


text:
COVID-19 Changes: Event listings come from various community organizations across Rye. Many are still in the process of reflecting new virtual events as well as the cancellation or postponement of in-person events.
If you are a community organization and would like to post events on MyRye.com, please get in touch.
- This event has passed.
Baby-wearing dance class
July 11, 2020 @ 2:00 pm - 2:30 pm
Join Ms. Sam, a certified GroovaRoo babywearing dance instructor, on Zoom for a private babywearing dance class. Wear comfy clothes and your favorite babywearing device. Don’t forget your water! Register to receive Zoom link. Contact: Samantha Mairson : 9142313162 : firstname.lastname@example.org

domain: artistic


2012 devinli biophysj .pdf
Original filename: 2012_devinli_biophysj.pdf
Title: The Molecular Mechanism Underlying Mechanical Anisotropy of the Protein GB1
Author: Yongnan Devin Li
This PDF 1.7 document has been generated by Elsevier / Acrobat Distiller 8.1.0 (Windows), and has been sent on pdf-archive.com on 13/12/2012 at 05:32, from IP address 24.87.x.x.
The current document download page has been viewed 1064 times.
File size: 633 KB (8 pages).
Privacy: public file
Download original PDF file
2012_devinli_biophysj.pdf (PDF, 633 KB)
Share on social networks
Link to this file download page
Biophysical Journal Volume 103 December 2012 2361–2368
The Molecular Mechanism Underlying Mechanical Anisotropy of the
Yongnan Devin Li,† Guillaume Lamour,†‡ Jo¨rg Gsponer,‡ Peng Zheng,† and Hongbin Li†*
Department of Chemistry and ‡Center for High-Throughput Biology, University of British Columbia, Vancouver, British Columbia, Canada
ABSTRACT Mechanical responses of elastic proteins are crucial for their biological function and nanotechnological use.
Loading direction has been identified as one key determinant for the mechanical responses of proteins. However, it is not clear
how a change in pulling direction changes the mechanical unfolding mechanism of the protein. Here, we combine protein engineering, single-molecule force spectroscopy, and steered molecular dynamics simulations to systematically investigate the
mechanical response of a small globular protein GB1. Force versus extension profiles from both experiments and simulations
reveal marked mechanical anisotropy of GB1. Using native contact analysis, we relate the mechanically robust shearing
geometry with concurrent rupture of native contacts. This clearly contrasts the sequential rupture observed in simulations for
the mechanically labile peeling geometry. Moreover, we identify multiple distinct mechanical unfolding pathways in two loading
directions. Implications of such diverse unfolding mechanisms are discussed. Our results may also provide some insights for
designing elastomeric proteins with tailored mechanical properties.
Mechanical properties of proteins are important not only for
their functions in biology (1) but also for opening new
possibilities in their use as building blocks in novel designer
biomaterials (2). Single-molecule force spectroscopy (3–5),
in concert with steered molecular dynamics (SMD) simulations (6–8), has greatly expanded our knowledge of the
design principles of mechanically stable proteins. Previous
work has shown that native topologies (7–9) and detailed
interactions, such as hydrophobic packing in protein structures (10,11), play important roles in determining protein
mechanical stability (12,13). In addition, mechanical
response of proteins to a stretching force is anisotropic
and depends on the loading direction (14–16). Two early
single-molecule atomic force microscopy (AFM) studies
(14,15) have shown the mechanical stability of globular
protein domains depends strongly on the linkage between
domains, which defines the loading direction. A mechanically stable protein can be surprisingly compliant when
stretched along a mechanically weak direction.
Because the loading direction is defined by the attachment points in a polymeric protein, researchers have developed several approaches (16–19) in controlling the linkage
chemistry. Using polyproteins (i.e., linear oligomers made
of monomeric domains covalently linked to each other) obtained with these techniques, researchers have demonstrated
a fascinating anisotropic mechanical response of individual
protein molecules to mechanical stress from different
loading directions (16,20). Recently, Marszalek and colleagues (21) explored the mechanical anisotropy of ankyrin
repeats by exploiting order of domain placements in the polSubmitted September 5, 2012, and accepted for publication October 26,
yprotein. Through the combination of single-molecule AFM
experiments and coarse-grained SMD simulations, these
authors are able to attribute this anisotropy to the different
ways in which native contacts in ankyrin are broken
between neighboring a-helices.
Despite significant progress in the understanding of
protein mechanical anisotropy, it is still unclear how
different pulling geometries, via change in the direction of
pulling force, alter the mechanical unfolding mechanism
of a protein. In light of this question, we attempt to explore
possible anisotropy in the mechanical response of a small
globular protein under an applied mechanical perturbation.
GB1, the B1 immunoglobulin G binding domain of streptococcal protein G, is chosen as the model protein due to its
small size and well characterized mechanical properties
(22,23). The mechanical unfolding mechanism of the 56
amino acid (aa) protein GB1 from its N- and C-termini
has been identified to be different from that of chemical
denaturant unfolding at the atomic level using SMD simulations (24–26). Recently, Graham and Best used a coarsegrained Go-like model to explore the switch of unfolding
pathways under a pulling force (27). They have found that
depending on the loading direction, the switch from the
intrinsic unfolding pathway to the novel mechanical
pathway can be either abrupt or gradual.
Here, we combine protein engineering, single-molecule
AFM experiments, and all-atom SMD simulations to
systematically investigate the anisotropic mechanical
response of GB1. Our experimental results support the
findings of Graham and Best (27), in which significant
anisotropy in the mechanical stability of GB1 is observed.
All-atom SMD simulations are used to gain insights into
the mechanical unfolding mechanism in each loading direction. We discuss the origin of such anisotropy based on
Editor: Daniel Muller.
Ó 2012 by the Biophysical Society
knowledge of protein structure and mechanical unfolding
MATERIALS AND METHODS
Gene encoding GB1 was generously provided by Dr. David Baker at the
University of Washington. Cysteine mutations on gene encoding GB1
were performed using the standard site-directed mutagenesis method and
subcloned into the expression vector pQE80L (QIAGEN, Valencia, CA).
All constructs were overexpressed in Escherichia coli strain DH5a and
purified using Co2þ affinity chromatography with TALON His-Tag purification resin (Clontech Laboratories, Mountain View, CA). The purified
protein samples (~2 mg/mL) were kept in phosphate-buffered saline
(PBS) at pH 7.4 containing 300 mM NaCl and 150 mM imidazole at
4 C. Names of the protein constructs contain two numbers, which are the
indices of the two cysteine residues (e.g., G10-40C).
Li et al.
All simulations were carried out at 300 K in the CHARMM22 (32) force
field with CMAP correction (33) using the implicit solvent FACTS (34).
The choice of the implicit solvent over explicit solvent was based on the
consideration of computational efficiency, as we wanted to carry out allatom simulations (param22 parameter file) and a rather large number of
trajectories needed to be collected. Calculations used an atom-based trun˚ , a nonbond cutoff of 12 A
˚ , and
cation scheme with a list cutoff of 14 A
˚ . Electrostatic interthe Lennard-Jones smoothing function initiated at 10 A
actions were force shifted. The SHAKE algorithm (35) was used for covalent bonds involving hydrogen atoms enabling integration time steps of 2 fs.
All native contact analyses were performed under VMD (36) using the
root mean-square deviation trajectory tool enhanced with a native contact
plug-in. Two atoms were considered in contact if their centers are within
˚ and only native contacts between heavy atoms were
a distance of 5 A
considered. For backbone atom-atom contacts, only Nitrogen and Oxygen
atoms from residues that are separated by at least two residues were considered. For side-chain atom-atom contacts, contacts between two adjacent
residues and within one residue were not taken into account.
Polyprotein construction through thiol-maleimide
RESULTS AND DISCUSSION
In a typical experiment, purified protein samples were concentrated to
~6 mg/mL using the Amicon Ultra-4 Centrifugal filter unit with Ultracel3 membrane (MILLIPOLE, Billerica, MA) and reacted with the chemical
cross-linker BM(PEO)3 (1,8-bis-maleimido-(PEO)3; Molecular Biosciences, Boulder, CO) as previously described (19). The solution was incubated at 37 C for ~8 h and stored at 4 C. Aliquots of the cross-linked
protein samples were used directly in AFM experiments.
It is well known that the shear topology, referring to the unfolding force being applied close to parallel to neighboring
b-strands joined by hydrogen bonds, is a critical criterion in
determining the mechanical stability of proteins. This places
requirements not only on the protein structure (i.e., native
topology) but also on the pulling axis (i.e., loading direction). In the context of this study, a pulling axis is defined
by two amino acid residues of the protein domain that serve
as the anchors. To design the pulling axes for probing the
mechanical anisotropy, we first looked at the three-dimensional structure of GB1. Structure of GB1 has been solved
at atomic resolution using both NMR spectroscopy (37)
and x-ray crystallography (38). The overall tertiary structure
of GB1 consists of a four stranded b-sheet packed against
a long a-helix, which belongs to the so called b-grasp
(39) or UB-roll (40) folding motif. The b-sheet can be
further broken into two structural elements, namely the
N-terminal and C-terminal b-hairpins (Fig. 1). Shearing
geometry enforced by the arrangement of terminal strands
constitutes the main point of mechanical resistance (9,24).
Graham and Best (27) have identified two classes of pulling
axes based on force-dependent unfolding kinetics from
simulation on GB1 using a coarse-grained Go-like model.
The mechanically strong class of pulling geometries has
their pulling axes aligned roughly along the long axis of
the b-sheet, whereas the mechanically weak class of axes
has their pulling axes lie between the b-sheet and the ahelix. We follow similar rationale in the design of pulling
axes (strong: G10-48C, G1-40C, and G19-56C where the
two numbers are indices of the anchoring residues);
however, due to technical caveats detailed in the Materials
and Methods section, it is challenging to work with the
mechanically weak class of axes identified in the study by
Graham and Best (27). Instead of axes lying between the
b-sheet and the a-helix, we choose pulling axes roughly
Single-molecule AFM experiments
Single-molecule AFM experiments were carried out on a custom-built
AFM constructed as described previously (28). Spring constants of the
Silicon Nitride cantilevers (MLCT, Bruker, Santa Barbara, CA) were calibrated in PBS before each experiment using the thermal noise method (29)
and typically had a value of ~50 pN/nm. In a typical experiment, polyprotein sample (~1 mL) was deposited onto a clean glass coverslip covered with
PBS (~50 mL) and was allowed to adsorb for ~5 min. Constant-velocity
AFM-pulling experiments were performed at 400 nm/s unless otherwise
noted. Contour length increment was calculated by subtracting initial
distance between the two tethered Ca atoms from the estimated distance
between the two in the stretched protein (estimated using: 0.36 nm/aa
number of aa between the two Ca atoms). Graham and Best (27) chose to
place one tethered Ca atom on the a-helix (residue number 32), which would
generally result in contour length increments much shorter than 10 nm
regardless of where the other tethered Ca atom was placed (residue 10 or
56). Relatively short polyprotein would create practical obstacles in AFM
˚ ), nonspeexperiments: although the spatial resolution of AFM is superb (~A
cific interactions are hard to avoid at short extension (<50 nm).
GB1, starting from the initial PDB structure 1PGA, was equilibrated for
1 ns during which it is reasonably stable, with Ca root mean-square devia˚ . The equilibrated final structure was used as the
tion in the range of 2 A
˚ /s) SMD pulling simulation perstarting point in constant velocity (0.1 A
formed with the AFM module of CHARMM (30,31). Each protein
construct was simulated by tethering Ca atoms at two amino acid positions
corresponding to the cysteine mutations. The protein was then subjected to
constant velocity stretching between the two tethered Ca atoms, in the
direction parallel to the line connecting the two at the beginning of the
Biophysical Journal 103(11) 2361–2368
Design of pulling axes
Mechanical Anisotropy of GB1
Based on these criteria, a total of five bicysteine constructs
were made (see Fig. 2).
Single-molecule AFM reveals anisotropic
response of GB1 to mechanical stress
FIGURE 1 Ribbon cartoon representation of the structure of GB1 (PDB:
1PGA) rendered with VMD (36). Bars between b-strands represent backbone hydrogen bonds between strand pairs. The b-strands are numbered
from the N-terminus to the C-terminus.
perpendicular to the long axis of the b-sheet (Fig. 2, G1948C and G10-40C).
In a previous study, we developed a protocol for constructing polyproteins with linkages between two precisely
controlled amino acid residue positions based on thiol-maleimide coupling chemistry (19). This method is similar to
the disulfide bond method developed by Rief and coworkers (16), which has been used to construct polyproteins. We want to emphasize that the loading direction in
AFM experiments is defined by the linkages in the polyprotein. In this approach, we need to mutate two native residues
in the protein into cysteine residues. Because native structure of GB1 lacks cysteine residues, concerns about formation of unwanted linkages are eliminated. Moreover, these
two native residues need to be solvent accessible and sufficiently far apart to avoid intramolecular linkage formation.
To investigate the effects of loading direction on the
mechanical unfolding of GB1, we carried out constantvelocity single-molecule force spectroscopy experiments
on each of the bicysteine GB1 constructs. The force-extension trace obtained from stretching a polyprotein of a
construct displays a characteristic sawtooth pattern (e.g.,
see bottom trace in Fig. 2 A). Each individual force peak
in the sawtooth pattern is the result of mechanical unfolding
of each individual domain in the polyprotein chain, except
for the last force peak, which corresponds to the extension
of the unfolded polyprotein and subsequent detachment
from either the AFM tip or substrate. Here, we want to
emphasize that mechanical unfolding of each domain
happens between the engineered cysteine residues. The
rising edge of each force peak is well described by the
worm-like chain model of polymer elasticity (41). Contour
length increments DLc calculated from a successive wormlike chain fit to the force peaks agree well with expected
values from structural considerations (see Materials and
Methods: single-molecule AFM experiment). These two
observations taken together suggest the folded protein
domains are completely unraveled between the points of
attachment in an all-or-none fashion.
Because mechanical unfolding of a protein is stochastic,
unfolding force of a protein will fluctuate randomly around
a mean value. The unfolding force histograms of the five
constructs at 400 nm/s pulling velocity are reported in
Fig. 2 B. G1-40C unfolds at a mean force of ~110 pN
making it the mechanically strongest construct of the five
FIGURE 2 Panel (A): Typical force versus
extension traces from single-molecule AFM experiments. Dashed lines are fits generated from an
interpolation formula of the worm-like chain
model (41). Typical persistence length used is
~0.4 nm. Panel (B): Normalized frequency histograms of unfolding force with Gaussian fits shown
as solid curves to guide the eye. Cartoon illustrations for the protein constructs are shown on the
right-hand side. Small spheres represent the Ca
atoms of the anchoring residues, whereas the
ribbon cartoons are based on the x-ray crystal
structure of GB1 (PDB:1PGA) (38).
Biophysical Journal 103(11) 2361–2368
pulling geometries we investigated, whereas the mechanically weakest G10-40C construct unfolds at ~40 pN. We
note that even the strongest construct unfolds at significantly
lower force than wt GB1, which unfolds at ~180 pN when it
is stretched from its N-C termini (22), suggesting that the
pulling direction along the N-C termini remains the most
mechanically resistant pulling geometry. From the unfolding force histograms, it is clear that the distribution of unfolding forces also depends strongly on the loading
direction. Furthermore, we observe that both the mean and
variance of unfolding forces are larger when the pulling
vector is close to parallel to the long axis of the b-sheet.
This is the case for constructs G1-40C, G19-56C, and
G10-48C. On the other hand, both values are smaller
when the pulling vector is not aligned with the long axis
of the b-sheet as seen in the histograms for constructs
G10-40C and G19-48C.
To further characterize the mechanical unfolding of GB1
under different loading geometries, we examined the dependency of the unfolding force on pulling velocity by
measuring the force extension relationships under different
pulling velocities. Because mechanical unfolding of all
constructs happens in an all-or-none fashion, the kinetics
of their mechanical unfolding can be modeled as a two-state
system with a force-dependent unfolding rate constant. The
Bell model (42) characterizes mechanical unfolding kinetics
using the unfolding rate constant at zero force a0 and the unfolding distance Dxu, which is the distance between the
native state and the mechanical unfolding transition state.
These two parameters can be extracted from pulling velocity
dependency data (Fig. 3) using a Monte Carlo simulation
based on a published protocol (43). From the values tabulated in Table 1, we note that pulling velocity dependency
of the unfolding force of G19-48C and G10-48C can be
Li et al.
TABLE 1 Mechanical unfolding data derived from constant
velocity single-molecule AFM experiments and Monte-Carlo
178 5 40
36 5 9
52 5 16
92 5 19
86 5 26
109 5 23
Fu denotes unfolding force and is reported as mean 5 SD (pulling velocity
is 400 nm/s). Dxu denotes the unfolding distance, which is the distance
between the native state and transition state of mechanical unfolding. a0
denotes the unfolding rate constant at zero force. Pulling velocity dependency for G10-40C was not examined due to low Fu close to the detection
limit of our AFM (~20 pN). Values for GB1 are taken from previously published results (22).
adequately reproduced using the same rate constant a0 as
GB1 but with a different unfolding distance Dxu. However,
a0 for GB1 fails to reproduce the behaviors of G19-56C and
G1-40C. For these constructs, both a0 and Dxu have
different values when compared to GB1. According to the
values of Dxu, the constructs can be divided into three
groups with small (GB1), intermediate (G1-40C, G1956C, and G10-48C), and large (G19-48C and possibly
G10-40C) Dxu values. A smaller Dxu value implies the transition state of mechanical unfolding is highly native-like.
Variations in the mean and variance of the unfolding force
reflect differences in the underlying mechanical unfolding
free energy profiles. The mean unfolding force is determined by the intrinsic unfolding rate constant at zero force
and the distance between the native and transition states
(Dxu). Because barrier crossing in mechanical unfolding is
thermal driven (hence stochastic), the variance/standard
deviation of the unfolding forces is governed by the relative
magnitude of the unfolding distance between folded and
transition states compared to thermal energy (kBT/Dxu).
Therefore, differences in unfolding kinetics/mechanism
will lead to changes in the mean and variance/standard deviation of the unfolding force distribution. It is reasonable to
anticipate that unfolding in mechanical strong (higher
mean value of unfolding forces) and weak (lower mean
value) directions proceed via different molecular mechanisms. It is of note that visual inspections on the five
constructs (Fig. 2) reveal that this shearing geometry is
roughly maintained in the cases of G10-48C, G19-56C,
and G1-40C; whereas G10-40C and G19-48C are arranged
to unfold the protein in a peeling or unzipping fashion.
SMD simulation provides insights into the
different mechanical unfolding mechanism
FIGURE 3 Dependency of unfolding force on the pulling velocity.
Kinetic parameters can be estimated from this dependency using Monte
Carlo simulations. Experimental unfolding forces for four constructs are
represented as symbols with error bars (mean 5 SD).
Biophysical Journal 103(11) 2361–2368
To learn about the mechanical unfolding mechanism for
each construct, we turned to SMD simulations. Results
from SMD studies have been shown to correlate well with
Mechanical Anisotropy of GB1
single-molecule AFM results (6,7,44). Previous results
(24–26,45) have indicated that terminal b-strands 1 and 4
of GB1 (Fig. 1) are in direct contact and form a mechanical
clamp motif that resists mechanical stress and protects the
protein from unfolding. Previous SMD simulations on
GB1 showed that the mechanical unfolding between its
N- and C-termini initiates with the separation of the terminal
b-strands 1 and 4 (24–26).
We first carried out similar SMD simulations on GB1,
which will serve as the benchmark for later comparisons.
One representative force-extension plot from such a simulation is shown in Fig. 4 F. The force peak at small extension
˚ ) is the main event in the graph, which corresponds
to the burst of the mechanical clamp. Fig. 4 F also shows
a snapshot of GB1 in the simulation trajectory right after
the burst. The snapshot shows that terminal b-strands 1
and 4 are separated from each other, whereas the C-terminal
hairpin is separated from the a-helix and N-terminal hairpin.
In some simulation trajectories (see Fig. 4 D1), there may
also be one or two minor force peaks, corresponding to
FIGURE 4 Force versus extension plots from constant velocity SMD
simulations. Gray traces are unsmoothed force versus extension traces
and black traces are the same traces smoothed with a moving median
(box size: 21 points). The common starting structure illustrated in Fig. 1
is not shown. Ribbon cartoon illustrating protein structures are taken just
after the main burst event, except for those in (A) and (B), which are taken
at an extension of ~12 A
unfolding intermediates not seen in experiments, following
the main force peak. All of the notable features of the simulations we have identified agree well with results from
previous studies that have been carried out with both
implicit and explicit solvent models (24–26). Such close
agreements suggest mechanical unfolding pathways identified from SMD simulations are robust and relatively insensitive toward differences between the chosen solvent
To directly compare between different loading directions
and elucidate the effects of loading directions on the
mechanical unfolding mechanism, we carried out constant
velocity SMD simulations for all of the protein constructs.
Typical force versus extension plots are shown in Fig. 4
along with a snapshot from each simulation trajectory. For
protein constructs G1-40C and G19-56C, we have included
two plots for each because there are two apparent unfolding
pathways. Even though unfolding force values obtained
from SMD simulations cannot be directly compared to the
experimental values due to the vast difference in timescale
of the two approaches, we note that our simulations results
correctly predict the relative rank of mechanical stabilities.
Indeed, our SMD simulations predict that the unfolding
force values will decrease from ~1.5 nN (for GB1) to ~1.2
nN (for G10-48C, G1-40C, and G19-56C) and finally to
nondetectable (for G10-40C and G19-48C), in excellent
agreement with experimental results.
Comparing Fig. 4, F and C, it clearly appears that G1048C unfolds through a pathway very similar to that of
GB1. However, force-extension plots for G1-40C and
C19-56C are much more complex. In their previous study
combining all-atoms and coarse-grained simulations (46),
West and colleagues have found that native interactions
are more important than nonnative contacts in determining
the origin of mechanical strength. Here, we monitor the fractions of native contacts between and within secondary structural elements (namely: N-terminal b-hairpin, C-terminal
b-hairpin, and a-helix) for each simulation trajectory. Backbone native contacts (i.e., backbone hydrogen bonds) within
structural elements are used as a gauge for the loss of
secondary structure; whereas side-chain native contacts
between structural elements as well as backbone hydrogen
bonds between the b-hairpins (i.e., formed between terminal
b-strands 1 and 4) are used to track the loss of tertiary
contacts. We have performed this analysis on all of the
protein constructs and the results are shown in Fig. 5.
Native contact analysis yields details in the
mechanical unfolding mechanism
In the case of GB1, native contact analysis reveals significant loss of native contact between C-terminal b hairpin
and a-helix during burst of the mechanical clamp at an
˚ . The burst of the mechanical clamp not
extension of ~5 A
only includes rupture of backbone hydrogen bonds between
Biophysical Journal 103(11) 2361–2368
Li et al.
FIGURE 5 Fraction of native contacts between
various structural elements of GB1 derived
from SMD simulations. Columns 1 to 7 contain
the fraction of intact native contacts between
various structural elements during the stretching
process: side-chain contacts between N- and
C-terminal b-hairpins (NþC hairpin), side-chain
contacts between N-terminal b-hairpin and a-helix
(N h.p.þhelix), side-chain contacts between
C-terminal b-hairpin and a-helix (C h.p.þhelix),
backbone contacts between N- and C-terminal bhairpins (NþC hairpin), backbone contacts within
N-terminal b-hairpin (N hairpin), backbone contacts within C-terminal b-hairpin (C hairpin), and
backbone contacts within the a-helix (helix).
Gray traces are derived from 10 individual SMD
simulations and the black trace is the average of
b-hairpins (Fig. 5 F4), but also includes substantial loss of
native contacts between the side chains (see Fig. 5, F1,
F2, F3). Following the main burst event, there are also
some disruptions to the backbone hydrogen bonds within
each structural element especially in the C-terminal
b-hairpin. The overall picture of the partially unfolded
GB1 from our SMD simulations involves rupture of backbone hydrogen bonds between b-hairpins and detachment
of the C-terminal b-hairpin from the core. This is in close
agreement with previous results (24–26).
Native contact analysis on simulation trajectories for
G10-48C (Fig. 5, C1, C2, C3, C4), shows the mechanical
unfolding pathway is very similar to that of wild-type
GB1, with some slight differences that may account for its
lower mechanical stability. Mechanical unfolding of G1048C is initiated with the concurrent rupture of backbone
hydrogen bonds between b-hairpins just like that of GB1.
However, both b-hairpins lose native contacts with the
a-helix at roughly the same rate, which clearly contrast the
asymmetry in the unfolding of GB1. There are some disruptions to the backbone hydrogen bonds in the C-terminal
b-hairpin, but these disruptions are quickly diminished
following the main burst event. The fact that the C-terminal
b-hairpin is not directly subject to mechanical stress for
G10-48C, unlike the case for GB1, is reflected in this
behavior. When compared to that of G10-48C, mechanical
unfolding of GB1 is a much more cooperative process that
causes disruption to almost all parts of the protein. Low
mechanical stability of G10-48C is also in accordance with
a conclusion drawn in our previous study where neighboring
b-strands were found to provide critical stabilization (47).
G1-40C and G19-56C are the other two constructs with
shearing geometry. SMD simulations on these constructs
show two distinct mechanical unfolding pathways for each
Biophysical Journal 103(11) 2361–2368
construct. In the case of G1-40C, both pathways differ
with that of GB1 in the sense that the N-terminal b-hairpin
is the one detached from the core. In one pathway (Fig. 4
E1), concurrent rupture of hydrogen bonds between b-hairpins is accompanied by sequential rupture of hydrogen
bonds within the N-terminal b-hairpin and the main burst
event. In the other pathway (Fig. 4 E2), hydrogen bonds
within the C-terminal b-hairpin are ruptured concurrently
along with sequential rupture of hydrogen bonds between
b-hairpins. Main burst events for both pathways occur at
a larger extension value than those of GB1 and G10-48C.
In the case of G19-56C, one mechanical unfolding pathway
(Fig. 4 D2) highly resembles that of GB1.The other pathway
(Fig. 4 D1) involves the concurrent rupture of hydrogen
bonds within the N-terminal b-hairpin accompanied by
sequential rupture of hydrogen bonds between b-hairpins.
G10-40C and G19-48C are the two constructs with
peeling geometry. SMD simulations show these constructs
unfold via similar pathways that involve the sequential
rupture of backbone hydrogen bonds. In the case of G1948C (Fig. 4 B), the C-terminal b-hairpin detaches from the
core like in the case of GB1. However, the peeling geometry
causes the backbone hydrogen bonds between b-hairpins to
rupture sequentially rather than concurrently. In the case of
G10-40C, sequential ruptures of backbone hydrogen bonds
happen within the C-terminal b-hairpin (Fig. 4 A). The
N-terminal b-hairpin detaches from the core like in the
case of G1-40C.
Diverse unfolding mechanisms lead to
anisotropic mechanical stability
In this study, we have combined single-molecule AFM and
SMD simulations to explore the anisotropic response of
Mechanical Anisotropy of GB1
a small globular protein to mechanical stress. Singlemolecule AFM experiments have revealed marked directional anisotropy in the mechanical response, whereas
SMD simulations have clarified its origin based on unfolding mechanisms. To the best of our knowledge, this is the
first time that mechanical anisotropy of a protein has been
systematically explained in detailed mechanistic terms
based on all-atom SMD simulations. Topological differences between shearing (G10-48C) and peeling (G19-48C)
geometries have been linked with mechanistic differences
between concurrent and sequential ruptures of critical backbone hydrogen bonds. This finding using only one protein is
in line with previous findings using two proteins with
different geometries (9).
Perhaps the most interesting finding from the SMD
simulations is the presence of parallel unfolding pathways.
Simulations reveal both G1-40C and G19-56C have two
apparently distinct unfolding pathways, whereas G10-48C
has only one. However, these three constructs display
similar behaviors in our experiments (in term of mean and
variance of unfolding force). Because the simulations are
carried out on a timescale that is much faster (~106)
compared to experiments, these possible parallel pathways
must be met with caution. A strategy involving the redesign
of unfolding pathways, which has been used in our previous
study (48), offers one method to test these predictions. In
such a scenario (e.g., take the case of G19-56C), a disulfide
bond (or a bihistidine metal binding site (49)) could be
engineered at an appropriate site across b-strands 1 and 2
to stabilize the N-terminal b-hairpin. This would result in
the GB1-like unfolding pathway (Fig. 4 D2) to be favored
and it could possibly shift the mean unfolding force to
a higher value.
Recent work (27) by Graham and Best is also concerned
with the role of force on the unfolding pathways of GB1. In
their coarse-grained G
o-like model based study, Graham
and Best have focused on the switch from an intrinsic unfolding pathway to a novel mechanical unfolding pathway
(27). The authors predict a nonlinear relationship between
the mean unfolding force and logarithm of the pulling
velocity for GB1 and G10-48C type constructs at very low
pulling velocity. This phenomenon has not been observed
in our experiments, possibly due to the relatively high pulling velocities used. Large differences between chemical and
mechanical unfolding rate constants at zero denaturant and
zero force (22) suggest that chemical and mechanical unfolding of GB1 follow different unfolding pathways, a result
consistent with our recent mechanical j-value analysis on
the mechanical unfolding of GB1 (50). It would be interesting to observe whether these two alternative pathways
do switch at low forces/pulling velocity. To do so, instrumental drift needs to be minimized and force detection limit
needs to be improved at the same time.
In our present study, we found that the small globular
protein GB1 exhibits clear directional anisotropy to
mechanical stress. The mechanically most robust construct
is the wild-type GB1 that unfolds at ~180 pN, whereas the
most labile construct is G10-40C that unfolds at ~40 pN.
Differences in unfolding mechanisms have been identified
to dictate differences in mechanical stability. Because
molecular determinants of mechanical stability are still
not completely understood (13), the rational design of
mechanical proteins and materials based on them will be
undoubtedly aided by detailed characterization of the unfolding pathways and effects of force on such pathways.
This work was supported by the Natural Sciences and Engineering
Research Council of Canada, Canada Foundation for Innovation and
Canada Research Chair program (to H.L.). Y.D.L. is partially supported
by an Alexander Graham Bell Canada Graduate Scholarship at the master’s
level from the Natural Sciences and Engineering Research Council of
1. Vogel, V. 2006. Mechanotransduction involving multimodular
proteins: converting force into biochemical signals. Annu. Rev.
Biophys. Biomol. Struct. 35:459–488.
2. Li, H., and Y. Cao. 2010. Protein mechanics: from single molecules to
functional biomaterials. Acc. Chem. Res. 43:1331–1341.
3. Rief, M., M. Gautel, ., H. E. Gaub. 1997. Reversible unfolding of
individual titin immunoglobulin domains by AFM. Science. 276:
4. Tskhovrebova, L., J. Trinick, ., R. M. Simmons. 1997. Elasticity and
unfolding of single molecules of the giant muscle protein titin. Nature.
5. Kellermayer, M. S. Z., S. B. Smith, ., C. Bustamante. 1997. Foldingunfolding transitions in single titin molecules characterized with laser
tweezers. Science. 276:1112–1116.
6. Lu, H., B. Isralewitz, ., K. Schulten. 1998. Unfolding of titin immunoglobulin domains by steered molecular dynamics simulation.
Biophys. J. 75:662–671.
7. Paci, E., and M. Karplus. 2000. Unfolding proteins by external forces
and temperature: the importance of topology and energetics. Proc.
Natl. Acad. Sci. USA. 97:6521–6526.
8. Klimov, D. K., and D. Thirumalai. 2000. Native topology determines
force-induced unfolding pathways in globular proteins. Proc. Natl.
Acad. Sci. USA. 97:7254–7259.
9. Carrion-Vazquez, M., A. F. Oberhauser, ., J. M. Fernandez. 2000.
Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering. Prog. Biophys. Mol. Biol. 74:63–91.
10. Ng, S. P., K. S. Billings, ., J. Clarke. 2007. Designing an extracellular
matrix protein with enhanced mechanical stability. Proc. Natl. Acad.
Sci. USA. 104:9633–9637.
11. Sadler, D. P., E. Petrik, ., D. J. Brockwell. 2009. Identification of
a mechanical rheostat in the hydrophobic core of protein L. J. Mol.
12. Forman, J. R., and J. Clarke. 2007. Mechanical unfolding of proteins:
insights into biology, structure and folding. Curr. Opin. Struct. Biol.
13. Crampton, N., and D. J. Brockwell. 2010. Unravelling the design principles for single protein mechanical strength. Curr. Opin. Struct. Biol.
14. Carrion-Vazquez, M., H. B. Li, ., J. M. Fernandez. 2003. The
mechanical stability of ubiquitin is linkage dependent. Nat. Struct.
Biophysical Journal 103(11) 2361–2368
15. Brockwell, D. J., E. Paci, ., S. E. Radford. 2003. Pulling geometry
defines the mechanical resistance of a beta-sheet protein. Nat. Struct.
16. Dietz, H., F. Berkemeier, ., M. Rief. 2006. Anisotropic deformation
response of single protein molecules. Proc. Natl. Acad. Sci. USA.
17. Yang, G. L., C. Cecconi, ., C. Bustamante. 2000. Solid-state synthesis
and mechanical unfolding of polymers of T4 lysozyme. Proc. Natl.
Acad. Sci. USA. 97:139–144.
18. Dietz, H., M. Bertz, ., M. Rief. 2006. Cysteine engineering of polyproteins for single-molecule force spectroscopy. Nat. Protoc. 1:80–84.
19. Zheng, P., Y. Cao, and H. B. Li. 2011. Facile method of constructing
polyproteins for single-molecule force spectroscopy studies. Langmuir.
20. Nome, R. A., J. M. Zhao, ., N. F. Scherer. 2007. Axis-dependent
anisotropy in protein unfolding from integrated nonequilibrium
single-molecule experiments, analysis, and simulation. Proc. Natl.
Acad. Sci. USA. 104:20799–20804.
21. Lee, W., X. Zeng, ., P. E. Marszalek. 2012. Mechanical anisotropy of
ankyrin repeats. Biophys. J. 102:1118–1126.
22. Cao, Y., C. Lam, ., H. Li. 2006. Nonmechanical protein can have
significant mechanical stability. Angew. Chem. Int. Ed. Engl. 45:
23. Cao, Y., and H. B. Li. 2007. Polyprotein of GB1 is an ideal artificial
elastomeric protein. Nat. Mater. 6:109–114.
24. Li, P. C., L. Huang, and D. E. Makarov. 2006. Mechanical unfolding of
segment-swapped protein G dimer: results from replica exchange
molecular dynamics simulations. J. Phys. Chem. B. 110:14469–14474.
25. Glyakina, A. V., N. K. Balabaev, and O. V. Galzitskaya. 2009. Mechanical unfolding of proteins L and G with constant force: similarities and
differences. J. Chem. Phys. 131:045102.
26. Glyakina, A. V., N. K. Balabaev, and O. V. Galzitskaya. 2009. Multiple
unfolding intermediates obtained by molecular dynamic simulations
under stretching for immunoglobulin-binding domain of protein G.
Open Biochem. J. 3:66–77.
27. Graham, T. G. W., and R. B. Best. 2011. Force-induced change in
protein unfolding mechanism: discrete or continuous switch? J. Phys.
Chem. B. 115:1546–1561.
28. Fernandez, J. M., and H. B. Li. 2004. Force-clamp spectroscopy monitors the folding trajectory of a single protein. Science. 303:1674–1678.
29. Florin, E. L., M. Rief, ., H. E. Gaub. 1995. Sensing specific molecular
interactions with the atomic force microscope. Biosens. Bioelectron.
30. Brooks, B. R., C. L. Brooks, 3rd, ., M. Karplus. 2009. CHARMM: the
biomolecular simulation program. J. Comput. Chem. 30:1545–1614.
Li et al.
33. Mackerell, Jr., A. D., M. Feig, and C. L. Brooks, 3rd. 2004. Extending
the treatment of backbone energetics in protein force fields: limitations
of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J. Comput.
34. Haberthu¨r, U., and A. Caflisch. 2008. FACTS: fast analytical
continuum treatment of solvation. J. Comput. Chem. 29:701–715.
35. Ryckaert, J. P., G. Ciccotti, and H. J. C. Berendsen. 1977. Numericalintegration of Cartesian equations of motion of a system with
constraints: molecular-dynamics of n-alkanes. J. Comput. Phys.
36. Humphrey, W., A. Dalke, and K. Schulten. 1996. VMD: visual molecular dynamics. J. Mol. Graph. 14:33–38, 27–28.
37. Gronenborn, A. M., D. R. Filpula, ., G. M. Clore. 1991. A novel,
highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science. 253:657–661.
38. Gallagher, T., P. Alexander, ., G. L. Gilliland. 1994. Two crystal
structures of the B1 immunoglobulin-binding domain of streptococcal
protein G and comparison with NMR. Biochemistry. 33:4721–4729.
39. Overington, J. P. 1992. Comparison of three-dimensional structures of
homologous proteins. Curr. Opin. Struct. Biol. 2:394–401.
40. Orengo, C. 1994. Classification of protein folds. Curr. Opin. Struct.
41. Marko, J. F., and E. D. Siggia. 1995. Stretching DNA. Macromolecules.
42. Bell, G. I. 1978. Models for the specific adhesion of cells to cells.
43. Carrion-Vazquez, M., A. F. Oberhauser, ., J. M. Fernandez. 1999.
Mechanical and chemical unfolding of a single protein: a comparison.
Proc. Natl. Acad. Sci. USA. 96:3694–3699.
44. Sotomayor, M., and K. Schulten. 2007. Single-molecule experiments
in vitro and in silico. Science. 316:1144–1148.
45. Cao, Y., and H. B. Li. 2008. Engineered elastomeric proteins with dual
elasticity can be controlled by a molecular regulator. Nat. Nanotechnol.
46. West, D. K., D. J. Brockwell, ., E. Paci. 2006. Mechanical resistance
of proteins explained using simple molecular models. Biophys. J.
47. Sharma, D., G. Feng, ., H. Li. 2008. Stabilization provided by neighboring strands is critical for the mechanical stability of proteins.
Biophys. J. 95:3935–3942.
48. Sharma, D., O. Perisic, ., H. Li. 2007. Single-molecule force spectroscopy reveals a mechanically stable protein fold and the rational
tuning of its mechanical stability. Proc. Natl. Acad. Sci. USA.
31. Brooks, B. R., R. E. Bruccoleri, ., M. Karplus. 1983. CHARMM:
a program for macromolecular energy, minimization, and dynamics
calculations. J. Comput. Chem. 4:187–217.
49. Cao, Y., T. Yoo, and H. B. Li. 2008. Single molecule force spectroscopy
reveals engineered metal chelation is a general approach to enhance
mechanical stability of proteins. Proc. Natl. Acad. Sci. USA.
32. MacKerell, A. D., D. Bashford, ., M. Karplus. 1998. All-atom empirical potential for molecular modeling and dynamics studies of proteins.
J. Phys. Chem. B. 102:3586–3616.
50. Shen, T., Y. Cao, ., H. Li. 2012. Engineered bi-histidine metal chelation sites map the structure of the mechanical unfolding transition state
of an elastomeric protein domain GB1. Biophys. J. 103:807–816.
Biophysical Journal 103(11) 2361–2368
Link to this page
Use the permanent link to the download page to share your document on Facebook, Twitter, LinkedIn, or directly with a contact by e-Mail, Messenger, Whatsapp, Line..
Use the short link to share your document on Twitter or by text message (SMS)
Copy the following HTML code to share your document on a Website or Blog


text:
Location: Third floor (five-story, elevator building), Isla Del Sol, Florida, Central West Coast. Near 25 miles of sandy beach.
Just minutes away from St. Petersburg Beach, Fort Desoto (voted one of the top five beaches in the United States). Two airports service this area (Tampa and St. Petersburg, Clearwater), 20-30 minutes from airport. Disney World and SeaWorld 90 minutes, Busch Gardens 30 minutes, and Sarasota 40 minutes. Easy driving to Kennedy Space Center.
Views of bay, heated pool, beach, hot tub, Don Cesar Resort, and golf course.


Very solid unit. Crisp look, came out of the box still kinda sharp around the edges, but itís nothing a grip or rail covers canít solve. If you leave it exposed, it looks amazing. One of JGís best made products, hands down.
Great build quality
This is an excellent rail system for the value. It is very nice quality aluminum, which adds almost no weight to your gun and does indeed free float the barrel. Easy to install and secure in place. If you're on a budget, and nowadays most of us are, this is a perfect way to upgrade your gun.


Summary of Minutes of the electronically conducted regular meeting of the City Commission held Monday, October 25, 2021, at 7:00 p.m.
Received petitions and communications.
Approved the following items on the Consent Calendar:
1) Minutes of the regular meeting of the City Commission held October 11, 2021.
2) Minutes of the closed session of the City Commission held October 11, 2021.
3) Bid of Tokio Marine HCC for 2022 stop loss coverage insurance.
4) S.A. 2-21 Resolution #4 to set a public hearing for November 8, 2021 on the PSD special assessment roll.
5) Updates to the PACT Policies as presented.
6) Warrants and Payrolls.
Held a public hearing and approved Resolution for transfer of the IFT from Dayco to MAC LTT as presented. (CC Exh. 13-2021)
Authorized the Mayor and Clerk to sign the IFT Agreement with MAC LTT as presented.
Approved a budget allocation from the Unassigned Fund Balance for the purchase and operation of an outdoor ice rink.
Approved a budget amendment from the Motor Pool Fund Balance for the repairs for the vactor truck.
Held a work session discussion on potential pedal trolley ordinance.
Adjourned the meeting at 8:49 p.m.


Traffic Information Access/Reuse Registration
Users must use this form to access traffic information from the Illinois Department of Transportation (IDOT) Gateway Traveler Information System (Gateway) for purposes such as republication or redistribution, inputting components of traffic information from other sources, or establishment of a link to this IDOT website. Gateway traffic information includes travel time and congestion data, camera images, dynamic message sign legend information, lane closure and construction information, and incident data. Gateway traffic information may be accessed via a periodic download of XML data. After submission of this form, registrants will be notified via Email whether the registration has been approved, and if so, detailed access instructions will be provided.
Note: Required fields are marked with an asterisk. Your password must be at least eight characters and contain characters from at least two of the following categories: lower case, upper case, digits, and symbols.


text:
Greyhound Trust Harvel is situated just outside the Kent village of Harvel, seven miles from the Gravesend A2 Tolgate Junction or three miles from Junction 3 of the M20 (Wrotham) and is run by Cheryl Miller with support from her staff.
We will be happy to introduce all of our dogs advising you on their background, temperament, likes and dislikes.
Our aim is to place these wonderful dogs in a safe environment where they can live life to the full, away from the confines of a kennel environment.
We offer full backup support whilst your new dog is settling in to home life and once settled we would be delighted to hear about their progress.
For further information please visit our website at this link : Harvel

domain: pet


text:
Our organization is 100% funded by donations. We are a volunteer based, non-profit cat rescue. Your donations will be put to great use helping to rescue and TNR cats in the Cambridge, Ontario area.
Learn more about Every Toebean Counts, our monthly donation programme and the benefit it will have to the homeless cats in the Cambridge, Kitchener and Waterloo area. Monthly donors will receive a tax receipt at the end of the year for all donations made during the year!
All donations over $20 will receive a tax receipt at the end of the year. 100% of all funds donated are used to help cats from supplies to medical issues and more.
We always have a need for litter, wet and dry food for kittens and adults. Anything you can send is very much appreciated!


text:
|Projects in Big Cities Remain A Major Hitch In EB-5 Debate
November 13, 2015
By Natalie Rodriguez
Law360, New York (November 13, 2015, 4:12 PM EST) -- With key EB-5 legislation set to expire in less than a month, talk of compromise on renewal legislation is heating up, but a debate over whether big-city projects will be effectively barred from taking advantage of the investment-fo-visa program remains a major stumbling block in talks, according to experts.
After getting a six0 week reprieve in September, legislation authorizing the EB-5 regional center program - which aids foreign investors in finding job-creating projects that qualify for the EB-5 green card incentive - is set to expire Dec. 11, and there has been a recent flurry of proposals aimed at forming legislation that might bring together opposing sides on the issues.

domain: law


text:
City to extend Centreville deal to 2022
TORONTO – The children’s amusement park on Toronto’s Centre Island will almost certainly remain in the hands of the company that has operated it for nearly five decades.
City staff will recommend council approve a renewal agreement with William Beasley Enterprises Ltd. that will give the company rights to operate Centreville until October 31, 2022. The deal gives the City an option to extend the agreement for an additional 10 years.
“We’re very happy,” says Bill Beasley, president of the family-owned Toronto company.
Financial terms are confidential until the proposal is before council on Wednesday. Beasley says the licensing fee his company pays to the City will be less but operating costs and capital investments will increase.
Ryan Glenn, manager of business services for the City of Toronto, says the new deal with Beasley includes “significant changes to the operation.”
Beasley will now be responsible for dredging the swan pond, maintaining grass, and paying for first-aid and water park attendants — previously responsibilities of the City.
The company has also committed to investing in improvements to the amusement park beginning in the 2014 season. Beasley says this includes replacing the Sky Ride gondola attraction, renovating food concessions, and adding new water features and a swing ride.
Beasley will also take over operation of Far Away Farm. “We’re looking forward to making it more educational and hands-on,” says Beasley.
At the end of the new lease, the operator will hand over certain assets to the City. (Glenn says this will level the playing field for future bids.) Beasley says the City can assume ownership of structures but his company will not hand over rides or the Centreville concept. “It’s our creation,” he says.
The City put out a request for proposals last September, nearly four months after its lease with Beasley expired.
Five companies downloaded the RFP document but only two submitted bids — Beasley and Billy G. Amusements Inc. of Mississauga. The latter’s proposal was declared non-compliant “as a result of a Mandatory Submission Requirement not being included.”
City staff decided Beasley “has demonstrated that it has the experience in all the areas necessary to successfully operate a children’s amusement area and the Far Enough Farm on Toronto Centre Island.”
Centreville is a collection of about 30 rides and food concessions that attracts as many as 750,000 visitors during its 111-day season.
Successful bidders for two additional contracts — one to operate food services on the Islands and one to manage the restaurant at the Centre Island ferry dock (currently a Shopsy’s) — have not yet been selected.
Those contracts are currently held by Beasley.

domain: economics


RÖSZKE, Hungary – Divisions over Europe’s refugee crisis deepened on Thursday, September 10 as record numbers of migrants streamed through the Balkans into Hungary, forcing Austria to suspend cross-border train services.
Germany, which is spearheading Europe’s response to the emergency, warned that an EU plan to distribute 160,000 new arrivals among member states was a mere “drop in the ocean”.
The plan already faces stiff opposition from eastern members, including those on the front line of the huge influx of people, who say they will not accept binding quotas from Brussels.
Foreign Minister Frank-Walter Steinmeier will try to win over eastern European states at a meeting in Prague on Friday, September 11.
The European Union is bitterly divided over how to cope with the biggest migrant crisis since World War II, with some countries on the bloc’s borders hardening their stance while others in Western Europe have opened their doors.
Some 22,500 refugees and migrants arriving on the Greek island of Lesbos have been registered by officials since Monday evening, September 7, according to police, many of them Syrian refugees setting sail from Turkey.
In Hungary, police said Thursday 3,321 people had entered in just 24 hours, hurrying to cross before tough new anti-migrant laws take effect, a new border fence is completed, and the weather worsens.
Austria’s train operator suspended services with Hungary on Thursday due to “massive overcrowding” and urged bus companies and volunteers to stop bringing migrants to stations.
In Serbia, where state television said 5,000 people had arrived at the border with Hungary, the prime minister warned the situation would worsen when new immigration laws come into force Tuesday, September 15.
‘An accountancy exercise’
The UN refugee agency on Tuesday, September 8 warned that at least 42,000 migrants were expected to enter Hungary by next week. (READ: Europe migrant crisis ‘manageable’ – UN refugee agency)
Many have endured treacherous sea journeys across the Mediterranean – most fleeing war and poverty in Syria, Afghanistan or Pakistan.
On Macedonia’s border with Greece, Agence France-Presse (AFP) journalists saw some 50 buses transporting some 2,500 migrants and 3 trains packed with 3,000 people departing from the town of Gevgelija.
EU interior ministers will meet Monday, September 14 to discuss how to share the burden across the bloc and ease the pressure on frontline states.
Germany, which has already welcomed 450,000 migrants this year, wants the 28-nation group to go further, calling for no limits to the quotas.
“The distribution of 160,000 refugees across Europe is a first step, if one wants to be polite,” said Deputy Chancellor Sigmar Gabriel. “It’s a drop in the ocean.”
But binding quotas are facing fierce resistance from eastern EU members.
“It is inappropriate to talk about mandatory quotas, calculated on an extremely bureaucratic basis, almost like an accountancy exercise I might say, without consulting member states,” said Romania President Klaus Iohannis.
His views echoed those of Slovakian Prime Minister Robert Fico, who said Wednesday he did not “want to wake up one day and have 50,000 people here about whom we know nothing”.
‘Walking for hours’
Juncker’s proposals include a possible revision of the EU’s much-criticized Dublin Treaty, under which asylum claims must be processed by the first country where refugees arrive.
EU lawmakers called for an international conference on migration bringing together the United States, United Nations and Arab countries.
Facing criticism that his government has been too slow to help, US President Barack Obama pledged to admit at least 10,000 Syrian refugees in the year starting October 1.
Spokesman Josh Earnest said Obama had ordered staff to “scale up” the number of Syrian refugee admissions from around 1,500 this year after over 62,000 Americans signed a petition calling on Washington to take in more people.
Meanwhile, on the Greek island of Lesbos, another flashpoint in Europe’s crisis, the boats kept arriving.
Hundreds – perhaps thousands – made a grueling 50-60 kilometer walk from their landing place to the main town where they must go to receive registration papers.
“We have been walking for 4 hours. There is no bus, no taxi, no water, no anything,” said Mohammed Yassin al-Jahabra, a 23-year-old English literature student.
Thousands of people have been forced to camp on the streets in squalid conditions, and there were repeated clashes as riot police struggled to control huge crowds pressing forward to get on board ferries.
But the boats are still arriving at an astonishing pace, with 6 landing in the space of an hour on Wednesday, AFP correspondents said.
“As soon as I put my feet down (on dry land), I stopped feeling tired,” said Feras Tahan, a 34-year-old Syrian graphic designer, his shoes and trousers soaked. – Sonia Bakaric, with Eric Randolph in Vienna, AFP/Rappler.com
There are no comments yet. Add your comment to start the conversation.


MARSEILLE (AFP) - French police arrested a man of Chechen origin on Saturday (May 19) who they said was exhibiting "strange" behaviour and carrying electrical equipment that could be used to make an explosive device.
The man was detained at the main railway station in the southern city of Marseilles after a member of the public reported he was acting bizarrely.
Police found suspicious electrical equipment on him and the man refused to give his name.
"What he had on him was not an explosive device, it was not dangerous in itself, but it was electrical, electronic equipment that could be used to make an explosive device," public prosecutor Xavier Tarabeux told Agence France-Presse.
"We do not yet know his identity but this man says he is Chechen and speaks Russian," he added.
France has been on high alert after a Chechen-born Frenchman stabbed a man to death last week and wounded four others in an attack in Paris that investigators believe was terror-inspired.
It was just the latest in a line of militant attacks that have killed more than 240 people in the last three years.
Police and prosecutors said they were interrogating the man arrested in Marseilles to ascertain whether he was planning an attack.
However, the city's mayor Jean-Claude Gaudin issued a statement saying an attack had been "foiled" and that the suspect had "obvious terrorist intentions".


text:
Discover the authentic Indonesian kitchen!
A warung – one of those small Indonesian eateries, where you are served the most fantastic, authentic dishes.
Orak Arek, Sayur Lodeh, Dagin Smoor – culinary gems that hail from the Emerald Belt. “Intensely flavoured dishes that we all love, but which so few places serve”, explains Marcel van Dulken. Luckily, they do sell them in the Vershal, at Warung Ventje. Selamat Makan!*
- Torenallee 36
- 5651 GW Eindhoven

domain: food


text:
We recommend you include the following information in your citation. Look below the item for additional data you may want to include.
Contact Owning Institution
All fields are required.
Duplication orders must be made through the Bancroft Library (UC Berkeley) request system, Aeon. Click the button below to order a copy of the item by signing up for an Aeon account or logging in to your existing account.
Six row seed planter. Each man plants about 50 plants per minute planting them in rows thirty-six inches apart and twenty-eight inches apart in the rows. An electric device on a corn planter check wire makes it possible to cross-cultivate both ways
The Bancroft Library. University of California, Berkeley

domain: library


text:
Trauma Resuscitation Unit
Jackson Health System has resumed normal operations at a majority of its facilities.
The UHealth Jackson Urgent Care at Cutler Bay is now open
The UHealth Jackson Urgent Care center at Keystone Point is now open
The UHealth Jackson Urgent Care center at Country Walk is now open
The Jefferson Reaves remain closed due to power outages.
To reschedule an appointment, please call 305-585-6000. Updated information for UHealth Jackson Urgent Care centers is available at JacksonUrgentCare.com.
Trauma Resuscitation Unit, Jackson Memorial Hospital
Emergency Room and Trauma Services
Albert Hernandez, ARNP, MSN
Senior Vice President and Chief Nursing Executive
Ric Cuming, RN, MSN, EdD, NEA-BC
- Ryder Trauma Center is the only Level One Adult Trauma Center in Miami-Dade County.
- Ryder Trauma Center is one of the 2 pediatric trauma centers in Miami-Dade County.
- It also has a receiving unit for all patients requiring Left Ventricular Assist Device (LVAD).
- Ryder Trauma Center has the only Level One Burn Trauma Center south of Orlando.
- We believe that those who are closest to patient care are the best participants for improving health care outcomes and the practice environment.
- Shared Governance Coordinating Council Presentation August 2011: Omnicell Optimization Project - A Focus on the JHS Pillars of Finance and Quality
Size & Type
The unit has 6 observation beds and 5 trauma bays.
22 RNs, 5 RNs certified to insert PICCs (Peripherally Inserted Central Catheters), 9 Emergency Services Care Associates (ESCA)
Staff Skill Mix
100% RN specifically trained in Trauma Nursing
Our organizational structure follows the Shared Governance model. It is made up of a nurse manager, associate nurse managers, clinical staff nurses, Emergency Services Care Associates (ESCA) and others.

domain: medical


text:
Author has written 1 story for Naruto.
I've decided to only post finished stories, so feel free to read my one-shots I have posted right now and review or PM!
Also, I have a lot of favorite stories, so feel free to browse through those if you're lacking reading material! :D
Some of them are just stunning, I would give them a try.
ALSO, if you have a request of any kind, feel free to send me a PM~! I would LOVE to attempt it.
|Focus:||Anime/Manga Skip Beat!|

domain: hobby


DraftSight has provided an affordable alternative to AutoCAD LT. I use DraftSight across all of our companies for IT, Engineering, and operations layouts. 90+% of the key commands used in AutoCAD work 1:1 in DraftSight, making the transition relativity easy.
Cost is the reason I went with DraftSight. I have been working with AutoCAD for most of my career, but it can be expensive. There is a free version, but I ended up paying the $300 for the 2-3 year renewal. Was well worth it!
90+% of the key commands used in AutoCAD work 1:1 in DraftSight, making the transition relativity easy.
When needing to collaborate with other departments, we were able to leverage the free version for small times and to avoid the expense of AutoCAD.
Some of our users had never used AutoCAD or a drafting software. DraftSight is laid out in an intuitive way that allowed some of our users to perform basic drawing modification on their own.
AutoCAD is a great solution, but very expensive for when you need a lightweight solution. DraftSight gave us the lightweight resources we needed but also was able to take on the big stuff when needed. DraftSight will provide more flexibility when needing to work on CAD drawings across an organization.


text:
Almost 51% polling stations categorised ‘sensitive’ by ECP
ISLAMABD (PEN) : Almost 51% of the polling stations have been categorised “sensitive” by the Election Commission of Pakistan for the upcoming general elections on February 8, on Monday.
Out of the total 90,675 polling stations, according to the electoral authority, 46,065 have been categorised as sensitive and 18,437 as highly sensitive.
ECP sources told that the total number of sensitive and most sensitive polling stations in Punjab has reached 18,620, and these include 12,580 sensitive and 6,040 most sensitive, whereas as many as 32,324 of the total 50,944 polling stations in the largest province have been declared as normal.
In Sindh, unlike Punjab, 5,937 out of the total 19,006 polling stations in the province have been categorised as normal. Of these, there are 6,545 sensitive and 6,524 most sensitive polling stations in the province.
Likewise, in Khyber Pakhtunkhwa, the number of sensitive and most sensitive polling stations comes to 10,309, and these include 6,166 sensitive and 4,143 highly sensitive ones. The total number of polling stations in the province is 15,697 out of which 5,388 are normal.
Quite understandably, the situation in Balochistan is a matter of serious concern in terms of security and sensitivity. Here, in this area-wise the largest but population-wise smallest province, the total number of polling stations is 5,028, out of which just 961 have been declared as normal and the remaining 4,067 are either sensitive or most sensitive. These include 2,337 sensitive and 1,730 most sensitive polling stations.
The ECP on Friday issued the final polling scheme for the February 8 general elections, envisaging a total of 90,675 polling stations with 2,76,402 polling booths to be set up across the country for over 128 million voters.
A senior official conceded that over 150,000 fewer polling booths are being established than required under the law, which will make it almost impossible at various places for all present to vote, if the turn-out is high.
As per the scheme, in Punjab, 50,944 polling stations will be established for the general elections, followed by 19,006 in Sindh, 15,697 in Khyber Pakhtunkhwa and 5,028 in Balochistan.
Section 59(6) of the Elections Act, 2017, requires publication of the final list of polling stations of each constituency at least 30 days ahead of the polling day in the official Gazette as well as on the website of the ECP. But the commission missed the legal deadline, it had set for itself of 15 days.
The polling scheme outlines the distribution of polling stations across all four provinces and the federal capital, categorising them as normal, sensitive, or highly sensitive based on the security situation and electoral violence history.
The ECP Thursday last, through a notification, said that unlike the 2018 general elections, the Army troops deployed for poll-related security duties will stay outside the polling stations for electoral exercise.

domain: news


text:
It was a last-minute plan but I was so glad it pushed through. For our second wedding anniversary celebration, we had a staycation at Azumi Boutique Hotel in Alabang. It was a newly-opened hotel, well-managed and beautiful in every aspect. We just had an overnight stay there but we enjoyed every minute of it. We checked into the hotel on Sunday and left the place Monday noon.
If you’re also planning for a staycation, the hotel is highly recommended. I heard their rooms are discounted until April 2014.
Here are some photos we took of our simple celebration:
Room 901. That’s the room where we stayed. It’s a corner room so we had a view of the busy Alabang-Zapote road outside.
The lobby was the hubby’s favorite part of the hotel so he made sure to have his picture taken there.
My shoes were killing my feet so I felt very relieved when I finally set them free. Aside from a queen bed, our room also has a day bed where one could just sit back and relax.
Soon after we settled into our room, we dozed off. When we woke up, we went up the Pent House where the infinity pool is located. We had it all to ourselves! But the water was cold and the wind was blowing hard we only got to swim for several minutes. Haha.
Who wouldn’t love an infinity pool with a great view of the Alabang skyline and Laguna de Bay?
We walked towards the nearby Molito to have dinner. We had ramen and gyoza at Ramen Yushoken. Good choice because we loved their food!
The Alabang skyline at night was breathtaking!
We loved the rainhead shower in our bathroom. For this alone, our splurge was worth it. Haha.
Romulo Cafe is the in-house restaurant of Azumi Boutique Hotel. On Monday morning, this was where we had our buffet breakfast. The selection was minimal but we enjoyed it, nonetheless.
We were on leave on Monday (the exact day of our anniversary!) so we were able to wake up late. By 11:00 am, we were already prepared to leave the hotel and head to Manila. I love this picture. We look so fresh and young here. Haha.
After we checked out of the hotel at around 12:00 noon, we spent the rest of the afternoon together running some errands and finally, joining the hubby’s family for dinner at Savory, SM Manila.
I still can’t believe we’ve made this far in our marriage. Two years is still two years. Here’s hoping the coming years in our married life will be as blissful and committed and blessed! 🙂
P.S. I also posted some collages on my IG account @edelweiss_19. Let’s follow each other there! 🙂
6 Replies to “Our 2nd Wedding Anniversary Celebration”
Of course, 2 years will take you to your 25th, 50th, to infinity and beyond. Invite nyo uli kami sa renewal of vows ah. 🙂 Happy Anniversary!
Sure, invite siyempre! Thanks Ella! 🙂
wow! nth honeymoon ba ito? happy anniversary to both of you! 🙂
Haha, may tama ka dyan. Thanks sis! 🙂
I love having staycations in Alabang. It’s like getting the best of both worlds – the fresh air you get when you go to a province and the “Manila vibe”.
Medyo late pero, HAPPY ANNIVERSARY! 🙂
Korek, presko pa nga ang hangin sa Alabang, parang samin din sa Bacoor. Thank you sis! 🙂

domain: travel


text:
Latest News (click title to open):
Primary Care Network (PCN)
A Primary Care Network (PCN) exists where GP practices work together at scale in a set community, with an aim to improve the care available to all patients living there. PCNs were first introduced into the NHS structure in 2019 there will usually be 30,000-50,000+ patients covered by a PCN. There are about 1,300 PCNs across the country. Each PCN is led by a Clinical Director.
Rothschild House Group is part of the Alpha PCN that covers Tring, Berkhamsted, Markyate areas, and also includes Manor Street Surgery and Gossoms End Surgery (both in Berkhamsted). We have collaborated for many years before the formation of PCNs, so in many respects the Alpha PCN created in 2019 is an extension of how we have always worked, for a combined patient population of over 50,000 people. The Clinical Director for Alpha PCN is Dr Pani Sissou, who is a Partner at Rothschild House Group.
Our vision is that we will work together both as practices, and with the wider health and social care system, to deliver safe, resilient, effective, equitable, high quality care to our Patient community.
The NHS provides additional funding direct to PCNs (but NOT to the individual GP practices) to support the service and contractual requirements of a PCN and to meet, or part meet, the costs of the PCN employing specifically allowed additional staff – this does not extend to additional GPs or Nurses. Some services, such as Care Home support or the provision of Extended/Enhanced Access (evening and weekend appointments) have or are being transferred by the NHS from a service provided by individual practices, to one provided by the PCN. Currently the following additional roles are provided through the PCN to both support our GPs with their workload and to widen the scope of services provided:
- Clinical Pharmacist
- First Contact Physio
- Health & Wellbeing Coach
- First Contact Mental Health Nurse
The PCN is invited each year to provide a series of health interventions and/or services, which include, but not limited to:
- Enhance Health in Care Homes – including regular reviews of care home patients;
- Supporting Early cancer Diagnosis – promoting screening, and ensuring early referral and identification of cancer where possible;
- Prevention and Diagnosis of Cardiovascular Disease – such as heart attacks and stroke;
- Structure Medication Reviews for stated cohorts – ensuring all medication remains appropriate and safe
Of course it’s possible that we offer many of these options already – but by working with other practices we can protect and grow this provision in the future
For more information on Primary Care Networks – see HERE


text:
The changing seasonal moods of Switzerland’s Matterhorn
Joshua Earle shares his experience of travelling to Switzerland to photograph one of Europe's highest peaks in t wo contrasting seasons
AS A PHOTOGRAPHER I’ve always been drawn to mountain landscapes, as they provide the ideal dramatic background for a picture. Mountains also appeal to me purely as a travel experience: there’s something about how they make you feel small and yet also significant, in that moment, that I really enjoy. That’s what prompted me to photograph the Matterhorn during two different seasons. There are perhaps two or three lakes around the mountain where you can see its reflection in the water, and this is one of them: Stellisee near Zermatt. For the whole of the trip I took in summer I was chasing the sort of light you can see here, and the way one face of the mountain is lit up in the sunrise. I used a tripod to take the picture; that’s me you can see in the shot. The second photograph was taken the following year in late winter, so the ski season was almost over, and the landscape was very quiet. I’d been waiting for three days to take the shot. I had hiked in the snow up a slightly roundabout route to reach this spot, and then set up camp there. It’s not a camping experience I would recommend to beginners. Temperatures were around -8°C and I needed a sturdy spade to dig the small trench that I set up the tent in. When you do this, it’s important to know the area well, so that you can be certain you’re setting up on solid ground. Because of my previous trip to Zermatt, I knew where I was and that it was safe
to set it up where I did. It was very difficult to tell: the snow so deep that you couldn’t even see the benches along the trail by the lake. While I waited for the cloud that often hugs the peak of the Matterhorn to clear, and for the sky to gain the luminous quality it has in this picture, I had the chance to quietly absorb the majesty of this vast, nearly deserted landscape. I find travelling alone a freeing experience, because it gives you the space to observe your surroundings without interruptions. When the right weather arrived, I took a picture of the mountain in the same way as I’d done the previous summer, intending the two images to work as a pair. I like both images, but the winter one particularly takes me back to a really special, inspiring experience and the beauty of the mountain.


text:
A Form 42 is an application for Appointment of Guardian Ad Litem, a person the court appoints to represent the best interests of a child or someone who is incompetent in a case. This form is used when a plaintiff in a case is an infant or an incompetent person that has no general or […]
Bankruptcy Lawyers in Charlotte, NC, Greensboro, NC, & Winston-Salem, NC
Duncan Law is a family owned law firm in Charlotte, NC, Greensboro, NC, and Winston-Salem, NC with areas of practice in bankruptcy, medical malpractice, nursing home abuse, and workers' compensation accidents. Whether you have questions with negligence issues, filing Chapter 13 bankruptcy or Chapter 7 bankruptcy, wrongful death actions, birth injuries, or any other legal issue, Terry Duncan in Charlotte, NC and Damon and Melissa Duncan, in Greensboro, NC will work hard to meet and exceed your expectations as we handle these very important issues.
Duncan Law was founded by Terry Duncan in 1996. Terry wished to create a law firm dedicated to representing those facing difficult times with bankruptcy, medical malpractice, nursing home abuse, serious injuries, and workers' compensation accidents.
Our website is designed to introduce you to our attorneys, but is also meant to provide you with simple and concise information about commonly asked question regarding bankruptcy, nursing home abuse, medical malpractice, workers' compensation and many other areas of law. Please browse our site and if you have any questions regarding an issue you want handled with dedication and toughness do not hesitate to contact an attorney at Duncan Law. By Damon Duncan
Duncan Law is dedicated to helping you and your family navigate through the difficult times of bankruptcy. Charlotte, NC bankruptcy lawyers, Greensboro, NC bankruptcy lawyers, and Winston-Salem, NC bankruptcy lawyers can answer your questions regarding the differences between Chapter 7 bankruptcy and Chapter 13 bankruptcy, describe the trustee, financial management counseling, and the courts, and address your concerns about your car, debts, mortgage, job, taxes, and loans.
Nursing Home Abuse is one of the most horrific legal problems you can face and Duncan Law takes it seriously. If a loved one has suffered broken bones from falls, serious infections, illness from wrong medications, malnutrition, serious bed sores, assaults by staff or other patients, unexplained hospitalization, or sexual assault while in a nursing or managed care home, you need legal help immediately. Your loved ones depend on you to protect them at their most vulnerable and Duncan Law wants to find you justice.
When a doctor, nurse, or hospital injures you or a loved one there is a legal remedy. Doctors and nurses are required to provide you with proper care during surgeries and your stay in a hospital. When they negligently fail in their actions, you need a lawyer that can ensure injuries don't go unnoticed. Medical malpractice can lead to serious problems such as birth injuries, cerebral palsy, misdiagnosis, cancer, serious surgical errors, and poor hospital care. These mistakes can have a profound impact on you or your loved ones' lives and the attorneys at Duncan Law want to reduce the amount of pain you might suffer.
An injury at work can have grave implications for your family and at Duncan Law we understand that your job is your life. When you have an injury at work, fall, hurt your back, arm, leg, shoulder, head, knee or ankle, are injured by machinery, develop carpal tunnel disease, suffer an occupational disease, or are attacked by a co-employee, it can keep you from providing for your family. North Carolina law provides specific benefits and your employer may be required to pay your medical bills and provide you and your family with benefits. Duncan Law wants to help ensure you receive all that you deserve.
We treat you as a person, not a case number. It is as simple as that. We are a firm that focuses on giving you the best legal service possible. That is what you deserve. To give great service, it is important to know the client. Your case is about more than laws and rules, it is about your story. We listen carefully to your story and work with you to reach the best results for your situation. We know that if we treat you right and give you the personal attention you deserve you will be more satisfied with your experience with Duncan Law.
Duncan Law's Recent Blog Posts
When it comes to filing a bankruptcy, one of the most frequently asked questions are “what should I tell my creditor’s after I have decided to file a bankruptcy?” One of the main issues that people with unsecured debt struggle with is the constant and harassing phone calls from creditors. In order to defer some […]
When you initially file a Chapter 13 bankruptcy your Trustee and creditors get a copy of a proposed plan. This tells the creditors how they are classified in the plan and how much you are estimating that they will be paid for the duration of the bankruptcy. Both the bankruptcy Trustee and the creditors have […]


text:
Dedicated to all the fancy, stylish, artsy and otherwise notable Latvians. Updates daily. I don't gain any kind of benefit from running this blog. Please message me if you want your work removed. Thank you for visiting and enjoy!
unbenannt by Reinis Traidas on Flickr.
Flying fashion by Arturs Tols on Flickr.
210 by Kriskjik on Flickr.
when cherry blooms (by Markus Riva)
CRW_8546 by Ethiopiate on Flickr.
Yo by Ethiopiate on Flickr.
228 by Kriskjik on Flickr.