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I traveled to Norway recently with Italian videographer/filmmaker and trekking guide Danilo Musetti, a member of renowned Italian photographer Luca Bracali’s film team, and was immediately struck by his artistry and adventurous, yet calm spirit. He was kind enough to agree to an interview and share more about himself and his work. He and his wife also operate a beautiful Tuscan inn (more information below). Thank you, Danilo!
How did you get into videography?
I have been a photographer since 1988. I traveled a lot and worked for many tourism boards taking photographsÂ for travel magazines. When digital photography came into being,Â I realized commercial photography would become more difficult — it became easy for anyone to get good results and prices dropped, so I decided to go into videography. I worked a lot with a friend (Pierluca Rossi) who produced travel stories for television. I’ve now beenÂ involved with video for about 17 years.
You’re going on a trekking expedition soon to Dolpo, Tibet, an area central to Peter Matthiessen’s “The Snow Leopard” correct?
Yes, I leave April 21 and returnÂ May 26. Iâ€™m going to the Upper Dolpo region (West Nepal) where Peter Matthiessen went to study blue sheep and look forÂ snow leopards.
See Danilo’s incredible filmÂ from a previous trip to Dolpo here:Â https://vimeo.com/197805736
Will you film there also?
Of course Iâ€™ll film the story as Iâ€™ve done in my 2009 and 2012 expeditions, but this time I’ll be trekking in spring, so the landscapes and the activities of the people will be very different. With this expedition Iâ€™ll be able to get an incredible amount of footage ofÂ the remote villages and â€œgompasâ€ (Tibetan Buddhist monasteries) of this incredible region where traditional Tibetan culture is still alive.
What do you love about that place?
TrekkingÂ in this region is a deep interior experience, no creature comforts, very high passes to climb, a difficult climate, and many hours ofÂ walking. But being there is like returning toÂ an era that seems unchangedÂ from 500 years ago. The Tibetan culture, the traditions and the very hard conditions of survival for the people who live there (very high mortality of children) transform this trip into a lifetime experience.
What’s your next big film project?
I have several projects coming up. For example, next year I want to film the â€œGreat Karakoram Traverseâ€ which will involve five weeks walking across one of the most spectacular mountain and glacierÂ routes and very close to the second highest mountain on earth, K2 in northernÂ Pakistan.
Your Tuscany Inn looks beautiful — is it open for booking year-round?Â
Yes, it is. I live in a part of the villa with my wife Monica and our daughter Viola Pilar. Because Iâ€™m a guide I organize activities like trekking, canyoning, caving, and mountain biking in the Apuan Alps and Appennino Tosco-Emiliano National Park mountains.
My wife Monica is very good cook and we can also offer guests a taste of traditional cuisine using local and high quality ingredients.
See the Inn’s website here:Â http://www.lalocandadelmoro.it/
If you could work anywhere in the world for a month, where would you go?
There are many places I like to visit, but because I climbed twice the volcano with a lava lake located in one of the hottest areas on earth ( Erta Ale in Dancalia depression, Ethiopia) I would like to climb the volcano with lava lake located in one of the coldest place on earth: Mt Erebus in Antartica.


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Another chemical that is affective as a urine odor remover. I previously wrote an article about the types of chemicals used as urine odor removers. Today I am going to add one more that is also used effectively at removing urine. These chemicals are useful for removing or eliminating many types of odors but we are specifically dealing with dog urine here so that is what I will address. I talk about 4 different types of chemicals in the other post. In this post I will describe a 5th type. It is an oxidizing agent. Many people ask me is there anything that really works on dog urine. The answer is yes. I want to point out three keys that will make the difference as to whether it does or does not work for you.
First: Use one of the types of chemicals that are effective at doing the job. There are many products being sold that are not even designed to remove the smell of urine. For example masking agents.
Second: Get a quality chemical. There are many chemicals designed to do the job but are of inferior quality. We all know about these.
Third: Use the chemical properly. This can be the biggest problem. For the job of urine odor removal you need to use the chemical as it was designed to be used or you will at best, only get partial urine odor removal. These chemicals must come in contact with the urine to be able to work on it. Some of them need a minimum amount of time in contact with the urine residue to work. They must be kept moist during that time. Some have temperature requirements to get results from them. These factors can vary some and you can still get partial results. But you need to keep these factors within the tolerances of the chemical you are using to get the complete results you want.
Oxidation happens to be one of the fastest methods of treatment. It is also excellent because it deals with all three aspects of urine deposits – odor, stain and contamination. The oxidation process releases large amounts of oxygen causing the urine to break down to more basic components like oxygen, nitrogen, carbon dioxide and other components. This process decontaminates the urine residue. In addition it removes the stain and neutralizes the odor. These basic components that are left then either leave as gases or are easily extracted and removed.

domain: chemistry


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The structural class information about a protein is important to understand its biological properties. NMR is one of the most powerful tools to obtain structural information of proteins in atomic resolution. However, an analysis of protein three-dimensional structure from NMR spectra usually requires laborious chemical shift assignment. We developed a new method for predicting the protein structural class directly from the NMR spectra without any chemical shift assignment. The results show that our method outperforms the methods using current secondary structure prediction.


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In a world where gaming on video game emulators has gained immense popularity 3ds emulators is just the icing on the cake. It allows you to play Nintendo console games on your PC by re-creating a platform which supports Nintendo console roms of the most popular games like Pokémon. Even though it is still in its early stages, it works fine with most games and is able to re-create the visual and audio output similar to the Nintendo console. It requires a PC or a laptop with high system requirements to operate on. Even though it can emulate a variety of software’s and programs but it is still not stable enough to play all the games.
Advantages and disadvantages of the Nintendo 3ds emulator
The Nintendo 3ds is a handheld portable gaming device. There are many emulators available for the 3ds ROMs to work on PC. The most famous of them is the Citra emulator which is an experimental open source 3ds emulator for emulating the Nintendo console games.
• It is the best 3ds emulator available online now.
• No extra software downloading is required to play. You just need to download the emulator only.
• It supports all the Nintendo console games including the region free 3ds system.
• As it is illegal to download commercial games, you cannot mention about this problem on any Citra online forums, or you will be instantly banned.
• All games are not playable on this emulator as because it is still in development phase.
• The original Nintendo 3ds Biosis required for the software to run and to copy the bios is illegal.
Why have Nintendo emulators gained so much in popularity?
There is an increasing popularity of the nintendo 3ds emulator because it can play and run most of the applications and games which were previously inaccessible to the PC users.

domain: hobby


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English Services provided:
Guide, Taxi driver, Host, Doctor, Lawyer, ask us any services About me: Shegby concierge bot (working 24/7) will help you if you can't find the Local person (or service) you need
Legendary street that is well-known worldwide, it’s also considered the busiest shopping spot in Europe. From large department store chains flagships and big multinational clothing shops to independent ones, Oxford Street has everything on its 1.5 miles (2.4km).
Museum Quarters in Vienna includes several museums with various expositions of modern arts. Here you always may find various festivals, concerts, expos. Inner yard of this complex is the favourite leisure place for city dwellers and Austrian capital guests.
This is a beachfront neighborhood of Honolulu that became famous mostly for its awesome beach area called Waikiki Beach. If you like surfing and any kind of beach activities, fun parties and cool restaurants you better see this place and stay there at least for a week.
Les Champs-Élysées is a central avenue and the main shop window of Paris running for about 2 km. Here you can see everything France is famous for: the Élysée Palace, monuments celebrating Napoleon's victories, world-famous boutiques, luxury hotels, beautiful fountains and graceful architecture.
Admiralty Wharf goes from the Palace Bridge up to the Senate Square along the shores of Neva river. Walk down and touch the copper lions – most famous lions in St.Petersburg. From the wharf you can see the Kunstkamera museum, the Palace and Annunciation Bridges.

domain: geography


WRIGHT TOWNSHIP — Wright Township police arrested five people on drug charges following a six-month investigation, authorities announced today.
The investigation began with a DUI traffic stop in August 2012. Over the course of the probe, police say six individuals sold marijuana and cocaine to an undercover member of the Luzerne County Drug Task Force in Wright Township and Warrior Run.
Wright Township police arrested Daniel Androckitis, 19, of Mountain Top; Matthew Edmonds, 23, of Lansford; Vanessa Kubik, 23, of Mountain Top; Kurt Smith, 19, of Mountain Top; and Damien Ward, 32, of Hazleton. Police have issued an arrest warrant for Joseph Estok, 21, of Mountain Top. He is expected to turn himself in upon his return to the area next week, police said.
With the exception of Estok, the suspects have each been arraigned before Magisterial District Judge Ronald Swank of Wright Township and charged with possession of a controlled substance, criminal use of communication facility, possession with the intent to deliver, criminal conspiracy and possession of drug paraphernalia.
Edmonds is incarcerated at Luzerne County Correctional Facility. Ward, Androckitis, Kubik and Smith have been released on bail.


Minsk Square: A tribute to east Europe in the middle of Bengaluru city
Bengaluru: For those visiting Bengaluru, after the stunning view of the Vidhan Soudha, it is the Minsk Square that is a head-turner. With a full-sized model of the Light Combat Aircraft (LCA) sitting majestically at the centre of the square, this busy square located next to Chinnaswamy Cricket Stadium is a famous landmark in the city. But unknown to many, the square is named after an Eastern European city – Minsk, capital of Belarus.
What is even more interesting is that the city Minsk has a square named after Bangalore. Even though many Soviet day statues and street names remain unchanged, a busy traffic junction and a huge park in Minsk was renamed after Bangalore, to show mutual respect.
The two cities, which are known as the silicon valleys of their respective countries, have a 35-year-long relationship. Even though forgotten or unknown to many Bengalureans, Minsk is a sister city of Bengaluru. The Sister City initiative between any two cities will be established to strengthen bilateral exchange relationships in the field of economy and trade, education, culture, information and technology and other sectors of municipal administration; and it dates back to post world war II era.
“Prior to this name change in 1989, Minsk Square was known as Queen’s Road Circle. Interestingly, then instead of the LCA fighter, the circle had a famous Ajeet fighter plane. The 1975-make single seater fighter-interceptor that was decommissioned in 1991 was installed at the centre of the junction,” recollected Wing Commander Prakash Kumar, a former Indian Air Force official who served in Bengaluru during that time.
Minsk, however, is not the only city with which Bengaluru shares the sister city tag. Bengaluru has signed similar MoUs with Reno City in Nevada and San Francisco in the United States of America. According to urban experts, apart from the MoU and naming circles (in the case of Minsk), these programmes have not made a big impact in improving the infrastructure and governance of the city.
A former IAS officer who served as the commissioner of the BBMP said that apart from junkets to these countries, the programme doesn’t boast of any innovations or changes. “In principle, this is a fantastic idea. The fact that a name like Minsk has become a household name in our city shows the success of the cultural exchange in the programme. But, in all three sister city projects, no considerable change on the ground has happened,” he said.
Urban expert V Ravichandar said that there are many issues with the programme itself. For example, who has the ownership of the city. “In Bengaluru BBMP is not an agency that has control over Bengaluru. We have Bescom, BDA and many other agencies. Unlike the mayor of Bengaluru city, a major of San Francisco has much higher authority. There is a big mismatch,” he pointed.
Ravichander also said that there is potential in the programme, but it is not being utilised. “To give an example, San Francisco has a wonderful open-source project, where all data related to city development is available in public. Developers and entrepreneurs can use this data to come up with solutions to the city’s problem. Even though our officers have visited San Francisco, we keep our data like a state secret. It is a lost opportunity,” he added.
However, it is not all a lost cost. In March this year, Minsk Major Vladimir Kukharev met with India’s Ambassador to the Republic of Belarus Sangeeta Bahadur to discuss ways of expanding cooperation between Minsk and Bengaluru. According to a statement, while the Indian side is interested in working in such areas as education, tourism, economy and joint ventures, and the Belarusian side is interested in IT, business and industry.
Following the meeting, both parties agreed to expand educational opportunities in technical specialities and information technology in Belarus for students from India. An ‘India Week’ is also expected to be held in Minsk in September 2022 to showcase the economic potential of the Belarusian capital for Indian guests, the statement added.
Meanwhile for the average Bengaluru, the “Minsk square” remains a part of their daily life, unaware of its connection with the former Soviet Union city.


Ada Integrated Environment III Computer Program Development Specification. Volume I. Ada Software Environment.
Interim rept. 15 Sep 80-15 Mar 81.
TEXAS INSTRUMENTS INC LEWISVILLE EQUIPMENT GROUP
Pagination or Media Count:
The Ada Integrated Environment AIE consists of a set of software tools intended to support design, developement and maintenance of embedded computer software. A significant portion of an AIE includes software systems and tools residing and executing on a host program computer or set of computers. This set is known as an Ada Programming Support Environment APSE. This B-5 Specification describes, in detail, the design for a minimal APSE, called a MAPSE. The MAPSE is the foundation upon which an APSE is built and will provide comprehensive support throughout the design, development and maintenance of Ada software. The MAPSE tools described in this specification include an Ada compiler, linkerloader, debugger, editor, and configuration management tools. The kernel KAPSE will provide the interfaces user, host, tool, database support, and facilities for executing Ada programs runtime support system. Author
- Computer Programming and Software


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Suspect arrested after allegedly holding towel over victim's face, knife to his throat in Lake Geneva hostage situation
LAKE GENEVA, Wis. (CBS 58) -- A suspect has been arrested after an alleged hostage situation in Lake Geneva.
Police were called out to the 100 block of West Street shortly after 4 a.m. on Sunday for a report of a man being held hostage with a knife to his throat.
Upon arrival, one of the residents of the home reported that the suspect was on the first floor of the home. As officers were setting up a perimeter, the suspect left the home with a knife in his hand. The suspect dropped the knife and fled on foot, jumping over a fence, and falling down an embankment.
Officers were able to take the suspect into custody.
He has been identified as Marc Fiorillo, 23, of Fontana.
One of the residents told police that he awoke to Fiorillo holding a towel over his face and a knife to his throat. The victim said that Fiorillo demanded drugs and money. One of the other residents of the home was able to call 911.
Police say that Fiorillo and the victims are not known to each other.
Fiorillo now faces charges of Armed Robbery with Use of Force, False Imprisonment, Burglary, and Resisting an Officer.

domain: news


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SINGAPORE: Forrest International, the corporate reputation
management company, has been appointed by English-language newspaper The
South China Morning Post.
Hong Kong-based SCMP has retained Forrest to provide strategic advice on
corporate positioning, investor relations and internal
The four-strong account team will be headed by board director Rosemary
Sayer, and will report direct to SCMP’s chairman Ean Kuok. The team will
include Leslie Fung, who joined the agency last month from the investor
relations team at Thomson Financial.
Fung also has a brief to work with technology-based clients including
Fosson, Asia Online and the Pioneer Global Group.
- Forrest is to promote a special event to raise the profile among Hong
Kong’s corporate leaders of Stanford University Business School, and its
executive education programmes.


A local father and son were being heralded as heroes on Tuesday after saving the life of their friend by performing CPR on him in West Yellowstone on Saturday.
The father and son, Rex and Blake Portmann, had just attended a CPR class two days before the incident.
Their friend, Tom Angell, needed CPR because he was having a heart attack.
Angell is an independent contractor and he used to run the Portmann's family ranch, the Diamond P.
This year, the Portmanns took control of operations again, and were helping Angell move his equipment out of their barn, when all of the sudden, he collapsed.
Blake Portmann watched Angell walk into his family barn, and go down.
"I just thought he fainted, and he didn't faint because we couldn't feel a pulse or anything like that, so we just immediately started doing CPR on him," Blake Portmann said.
Blake Portmann started doing chest compressions right away, and yelled for his dad, Rex Portmann.
"I saw what was happening, and then, all I know is, I was there. You know, I was here and there," Rex Portmann said.
Blake and Rex Portmann and Rex's son-in-law Charles traded off doing chest compressions for 20 minutes.
"And that was the longest 20 minutes I've ever waited in my life," Blake Portmann said.
Just 48 hours earlier, Rex and Blake Portmann were learning to do chest compressions for the first time in a long time.
"The last time I was CPR certified was in 1988, maybe '89?" Rex Portmann said.
The Portmanns' teacher, Sharra Nelson, said she had just passed out a hands-only CPR card to her class last Thursday night, and both of the men remembered that card when they started working. She said this incident is proof of how important it is to become CPR certified.
"Instead of just standing around waiting for the EMS to show up, we want you to get down and start doing compressions, because just the compressions only can save a life," Nelson said.
And in this case, it did.
"It was a simple class, it was three hours, but it was worth every second that I was there," Blake Portmann said.
After EMS showed up, Angell was airlifted to Eastern Idaho Regional Medical Center.
Angell is still in the hospital, but is now in stable condition, said Rex Portmann.
Both Portmanns work at Portneuf Medical Center. They were getting certified as part of their U.S. Forest Service permit for the guided horse tours they do at Diamond P Ranch in Yellowstone National Park.


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Stayed here for 7 nights with 2 other guests, I would have to say the apartment is small in comparison to other 1 bedroom apartments I have stayed in before, the only eating area was the table and three chairs on the balcony, there wasn't an indoor eating area, not even a breakfast bar. The pull down sofa bed was... More
- Reservation Options:
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- Aparthotel Buenos Aires Playa Del Ingles, Gran Canaria

domain: travel


Celebrity › Nicolas Cage
An Oscar award winner Nicolas Cage is one of the most successful actor of the Hollywood film industry. Having made his name with anxious, well-received portrayals of wild men and anti-heroes, actor Nicolas Cage underwent remarkable mid-career transformations, remaking himself as action star, romantic lead, and most recently, director and producer.
Regarded as one of the most multifaceted actors of our time, Nicolas Cage is equally well known for his passionate portrayals in drama and comedy. He earned an Academy Award and a Golden Globe Award, as well as Best Actor Awards from the New York Critics Circle, the Los Angeles Film Critics Association, the Chicago Film Critics and the National Board of Review for his performance as an alcoholic who drinks himself to death in "Leaving Las Vegas."
Nicolas took a job selling popcorn at the Fairfax Theatre, thinking that would be the only route to a movie career. However, he got a small part in his uncle's film 'Rumble Fish' in 1983. He was criticised for his over-the-top performance in 'Peggy Sue Got Married', but attracted the attention of Cher, who suggested him for a role in 'Moonstruck'. She even walked out of the production for a day, until the producers gave in to her demand, and 'Moonstruck' turned out to be his first really big box-office hit.
Nicolas Cage no doubt owes part of his success to his fabulous work imperative and cleverness; he goes all out in preparing for roles, from undergoing a tooth extraction without novocaine for Birdy (1984) to learning the mandolin for Captain Corellis Mandolin (2001). Nicolas Cage made his film debut in 1982's Fast Times at Ridgemont High , but it was his role in the hit Valley Girl (1984) that launched his screen career. Cage was suddenly a reliable star after 1987's Raising Arizona (with Frances McDormand) and Moonstruck (with Cher), known for his intensity and attachment for improvement on the set. He won an Oscar as the self-destroying drunk in Leaving Las Vegas in 1995, and by the end of the decade was starring in big-budget action movies, including The Rock (1996, with Sean Connery), Con Air (1997, with Ving Rhames), Face/Off (1997, opposite John Travolta) and Gone in Sixty Seconds (2000, with Angelina Jolie). In 2003 he was again Oscar-nominated for playing nervous screenwriter Charlie Kaufman in the offbeat drama Adaptation.


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Located within the heart of a devastated city, Mercy Square is an over-populated, rubble walled settlement of urban dwelling, post apocalyptic survivors. Founded by Mayor John Greenburg (Area 4) and Sheriff Logan Bolton (Area 4), this community was based on the aspects of hope, safety, and restoration of civilization. Since the community’s humble beginnings of a mere sixty some citizens, the secured urban area now houses over a thousand survivors. Although the buildings within the complex are perfectly capable of providing the needs of shelter for at least another five hundred or more, the overall feeling of this urban safe-hold is that the community is bursting at the seams.
In essence, Mercy Square is suffering from something that is a hard found blessing in the ruins of the apocalypse. That combination of a blessing and a curse is overpopulation. While the community offers the perfect model for the rebirth of civilization as it was once known, the ruins of the surrounding metropolitan city is quickly running dry in regards to needed resources. The community has a constant need for food, fresh water, and gasoline. While the community’s scouts, scavengers, and drivers make a constant effort to supply the community’s needs in recovered goods, such ventures are starting to fall short of need.

domain: sociology


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The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient's ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.
24 Related JoVE Articles!
Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching
Institutions: University of Rochester, University of Rochester.
Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) of macromolecules, and can be applied to both in vitro
and in vivo
biological systems. Multi-fluorescence recovery after photobleaching is performed by photobleaching a region of interest within a fluorescent sample using an intense laser flash, then attenuating the beam and monitoring the fluorescence as still-fluorescent molecules from outside the region of interest diffuse in to replace the photobleached molecules. We will begin our demonstration by aligning the laser beam through the Pockels Cell (laser modulator) and along the optical path through the laser scan box and objective lens to the sample. For simplicity, we will use a sample of aqueous fluorescent dye. We will then determine the proper experimental parameters for our sample including, monitor and bleaching powers, bleach duration, bin widths (for photon counting), and fluorescence recovery time. Next, we will describe the procedure for taking recovery curves, a process that can be largely automated via LabVIEW (National Instruments, Austin, TX) for enhanced throughput. Finally, the diffusion coefficient is determined by fitting the recovery data to the appropriate mathematical model using a least-squares fitting algorithm, readily programmable using software such as MATLAB (The Mathworks, Natick, MA).
Cellular Biology, Issue 36, Diffusion, fluorescence recovery after photobleaching, MP-FRAP, FPR, multi-photon
Characterization of Thermal Transport in One-dimensional Solid Materials
Institutions: Iowa State University.
The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials, including conductive, semi-conductive or nonconductive one-dimensional structures. This technique broadens the measurement scope of materials (conductive and nonconductive) and improves the accuracy and stability. If the sample (especially biomaterials, such as human head hair, spider silk, and silkworm silk) is not conductive, it will be coated with a gold layer to make it electronically conductive. The effect of parasitic conduction and radiative losses on the thermal diffusivity can be subtracted during data processing. Then the real thermal conductivity can be calculated with the given value of volume-based specific heat (ρcp
), which can be obtained from calibration, noncontact photo-thermal technique or measuring the density and specific heat separately. In this work, human head hair samples are used to show how to set up the experiment, process the experimental data, and subtract the effect of parasitic conduction and radiative losses.
Physics, Issue 83, thermal transport, thermal diffusivity, thermal conductivity, transient electro-thermal technique, volume-based specific heat, human head hair
Scalable Nanohelices for Predictive Studies and Enhanced 3D Visualization
Institutions: University of California Merced, University of California Merced.
Spring-like materials are ubiquitous in nature and of interest in nanotechnology for energy harvesting, hydrogen storage, and biological sensing applications. For predictive simulations, it has become increasingly important to be able to model the structure of nanohelices accurately. To study the effect of local structure on the properties of these complex geometries one must develop realistic models. To date, software packages are rather limited in creating atomistic helical models. This work focuses on producing atomistic models of silica glass (SiO2
) nanoribbons and nanosprings for molecular dynamics (MD) simulations. Using an MD model of “bulk” silica glass, two computational procedures to precisely create the shape of nanoribbons and nanosprings are presented. The first method employs the AWK programming language and open-source software to effectively carve various shapes of silica nanoribbons from the initial bulk model, using desired dimensions and parametric equations to define a helix. With this method, accurate atomistic silica nanoribbons can be generated for a range of pitch values and dimensions. The second method involves a more robust code which allows flexibility in modeling nanohelical structures. This approach utilizes a C++ code particularly written to implement pre-screening methods as well as the mathematical equations for a helix, resulting in greater precision and efficiency when creating nanospring models. Using these codes, well-defined and scalable nanoribbons and nanosprings suited for atomistic simulations can be effectively created. An added value in both open-source codes is that they can be adapted to reproduce different helical structures, independent of material. In addition, a MATLAB graphical user interface (GUI) is used to enhance learning through visualization and interaction for a general user with the atomistic helical structures. One application of these methods is the recent study of nanohelices via MD simulations for mechanical energy harvesting purposes.
Physics, Issue 93, Helical atomistic models; open-source coding; graphical user interface; visualization software; molecular dynamics simulations; graphical processing unit accelerated simulations.
Echo Particle Image Velocimetry
Institutions: University of New Hampshire.
The transport of mass, momentum, and energy in fluid flows is ultimately determined by spatiotemporal distributions of the fluid velocity field.1
Consequently, a prerequisite for understanding, predicting, and controlling fluid flows is the capability to measure the velocity field with adequate spatial and temporal resolution.2
For velocity measurements in optically opaque fluids or through optically opaque geometries, echo particle image velocimetry (EPIV) is an attractive diagnostic technique to generate "instantaneous" two-dimensional fields of velocity.3,4,5,6
In this paper, the operating protocol for an EPIV system built by integrating a commercial medical ultrasound machine7
with a PC running commercial particle image velocimetry (PIV) software8
is described, and validation measurements in Hagen-Poiseuille (i.e.
, laminar pipe) flow are reported.
For the EPIV measurements, a phased array probe connected to the medical ultrasound machine is used to generate a two-dimensional ultrasound image by pulsing the piezoelectric probe elements at different times. Each probe element transmits an ultrasound pulse into the fluid, and tracer particles in the fluid (either naturally occurring or seeded) reflect ultrasound echoes back to the probe where they are recorded. The amplitude of the reflected ultrasound waves and their time delay relative to transmission are used to create what is known as B-mode (brightness mode) two-dimensional ultrasound images. Specifically, the time delay is used to determine the position of the scatterer in the fluid and the amplitude is used to assign intensity to the scatterer. The time required to obtain a single B-mode image, t
, is determined by the time it take to pulse all the elements of the phased array probe. For acquiring multiple B-mode images, the frame rate of the system in frames per second (fps) = 1/δt
. (See 9 for a review of ultrasound imaging.)
For a typical EPIV experiment, the frame rate is between 20-60 fps, depending on flow conditions, and 100-1000 B-mode images of the spatial distribution of the tracer particles in the flow are acquired. Once acquired, the B-mode ultrasound images are transmitted via an ethernet connection to the PC running the PIV commercial software. Using the PIV software, tracer particle displacement fields, D(x,y)
[pixels], (where x and y denote horizontal and vertical spatial position in the ultrasound image, respectively) are acquired by applying cross correlation algorithms to successive ultrasound B-mode images.10
The velocity fields, u(x,y)
[m/s], are determined from the displacements fields, knowing the time step between image pairs, ΔT
[s], and the image magnification, M
. The time step between images ΔT
= 1/fps + D(x,y)/B
, where B
[pixels/s] is the time it takes for the ultrasound probe to sweep across the image width. In the present study, M = 77[μm/pixel], fps
= 49.5[1/s], and B
= 25,047[pixels/s]. Once acquired, the velocity fields can be analyzed to compute flow quantities of interest.
Mechanical Engineering, Issue 70, Physics, Engineering, Physical Sciences, Ultrasound, cross correlation, velocimetry, opaque fluids, particle, flow, fluid, EPIV
Designing Silk-silk Protein Alloy Materials for Biomedical Applications
Institutions: Rowan University, Rowan University, Cooper Medical School of Rowan University, Rowan University.
Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi
) and domestic mulberry silk (Bombyx mori
) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.
Bioengineering, Issue 90, protein alloys, biomaterials, biomedical, silk blends, computational simulation, implantable electronic devices
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling
Institutions: Colorado State University, USDA-ARS, Colorado State University.
Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2
fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13
C with 15
O or 2
H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4
. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e
. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7
. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13
C and 15
N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13
C and 15
N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii
Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13
C and 6.7 atom%15
N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13
C and 0.56 atom%15
N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2
concentration, and light levels in an airtight 13
atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.
Environmental Sciences, Issue 83, 13C, 15N, plant, stable isotope labeling, Andropogon gerardii, metabolic compounds, structural compounds, hot water extraction
Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+
release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis
Institutions: Virginia Tech, Virginia Tech.
Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process.
cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles.
Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).
Biomedical Engineering, Issue 79, Medicine, Cellular Biology, Molecular Biology, Bioengineering, Anatomy, Physiology, Biophysics, Physics, Microfluidics, Cell Separation, Microfluidic Analytical Techniques, Electrophoresis, Microchip, cancer diagnosis, cell enrichment, cell sorting, microfluidics, dielectrophoresis, Lab on a chip, cells, imaging
High-speed Particle Image Velocimetry Near Surfaces
Institutions: University of Michigan.
Multi-dimensional and transient flows play a key role in many areas of science, engineering, and health sciences but are often not well understood. The complex nature of these flows may be studied using particle image velocimetry (PIV), a laser-based imaging technique for optically accessible flows. Though many forms of PIV exist that extend the technique beyond the original planar two-component velocity measurement capabilities, the basic PIV system consists of a light source (laser), a camera, tracer particles, and analysis algorithms. The imaging and recording parameters, the light source, and the algorithms are adjusted to optimize the recording for the flow of interest and obtain valid velocity data.
Common PIV investigations measure two-component velocities in a plane at a few frames per second. However, recent developments in instrumentation have facilitated high-frame rate (> 1 kHz) measurements capable of resolving transient flows with high temporal resolution. Therefore, high-frame rate measurements have enabled investigations on the evolution of the structure and dynamics of highly transient flows. These investigations play a critical role in understanding the fundamental physics of complex flows.
A detailed description for performing high-resolution, high-speed planar PIV to study a transient flow near the surface of a flat plate is presented here. Details for adjusting the parameter constraints such as image and recording properties, the laser sheet properties, and processing algorithms to adapt PIV for any flow of interest are included.
Physics, Issue 76, Mechanical Engineering, Fluid Mechanics, flow measurement, fluid heat transfer, internal flow in turbomachinery (applications), boundary layer flow (general), flow visualization (instrumentation), laser instruments (design and operation), Boundary layer, micro-PIV, optical laser diagnostics, internal combustion engines, flow, fluids, particle, velocimetry, visualization
Quantifying Mixing using Magnetic Resonance Imaging
Institutions: University of California, Davis, Procter & Gamble Company.
Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1
. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2
. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1
H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2
). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.
Biophysics, Issue 59, Magnetic resonance imaging, MRI, mixing, rheology, static mixer, split-and-recombine mix
Magnetic Resonance Derived Myocardial Strain Assessment Using Feature Tracking
Institutions: Cincinnati Children Hospital Medical Center (CCHMC), Imaging Systems GmbH, Advanced Medical Imaging Development SRL, The Christ Hospital.
Purpose: An accurate and practical method to measure parameters like strain in myocardial tissue is of great clinical value, since it has been shown, that strain is a more sensitive and earlier marker for contractile dysfunction than the frequently used parameter EF. Current technologies for CMR are time consuming and difficult to implement in clinical practice. Feature tracking is a technology that can lead to more automization and robustness of quantitative analysis of medical images with less time consumption than comparable methods.
Methods: An automatic or manual input in a single phase serves as an initialization from which the system starts to track the displacement of individual patterns representing anatomical structures over time. The specialty of this method is that the images do not need to be manipulated in any way beforehand like e.g. tagging of CMR images.
Results: The method is very well suited for tracking muscular tissue and with this allowing quantitative elaboration of myocardium and also blood flow.
Conclusions: This new method offers a robust and time saving procedure to quantify myocardial tissue and blood with displacement, velocity and deformation parameters on regular sequences of CMR imaging. It therefore can be implemented in clinical practice.
Medicine, Issue 48, feature tracking, strain, displacement, CMR
Experimental Measurement of Settling Velocity of Spherical Particles in Unconfined and Confined Surfactant-based Shear Thinning Viscoelastic Fluids
Institutions: The University of Texas at Austin.
An experimental study is performed to measure the terminal settling velocities of spherical particles in surfactant based shear thinning viscoelastic (VES) fluids. The measurements are made for particles settling in unbounded fluids and fluids between parallel walls. VES fluids over a wide range of rheological properties are prepared and rheologically characterized. The rheological characterization involves steady shear-viscosity and dynamic oscillatory-shear measurements to quantify the viscous and elastic properties respectively. The settling velocities under unbounded conditions are measured in beakers having diameters at least 25x the diameter of particles. For measuring settling velocities between parallel walls, two experimental cells with different wall spacing are constructed. Spherical particles of varying sizes are gently dropped in the fluids and allowed to settle. The process is recorded with a high resolution video camera and the trajectory of the particle is recorded using image analysis software. Terminal settling velocities are calculated from the data.
The impact of elasticity on settling velocity in unbounded fluids is quantified by comparing the experimental settling velocity to the settling velocity calculated by the inelastic drag predictions of Renaud et al.1
Results show that elasticity of fluids can increase or decrease the settling velocity. The magnitude of reduction/increase is a function of the rheological properties of the fluids and properties of particles. Confining walls are observed to cause a retardation effect on settling and the retardation is measured in terms of wall factors.
Physics, Issue 83, chemical engineering, settling velocity, Reynolds number, shear thinning, wall retardation
Magnetic Tweezers for the Measurement of Twist and Torque
Institutions: Delft University of Technology.
Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a “conventional” magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the “conventional” magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.
Bioengineering, Issue 87, magnetic tweezers, magnetic torque tweezers, freely-orbiting magnetic tweezers, twist, torque, DNA, single-molecule techniques
The Preparation of Electrohydrodynamic Bridges from Polar Dielectric Liquids
Institutions: Wetsus - Centre of Excellence for Sustainable Water Technology, IRCAM GmbH, Graz University of Technology.
Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and polar dielectric liquids. These bridges are unique from capillary bridges in that they exhibit extensibility beyond a few millimeters, have complex bi-directional mass transfer patterns, and emit non-Planck infrared radiation. A number of common solvents can form such bridges as well as low conductivity solutions and colloidal suspensions. The macroscopic behavior is governed by electrohydrodynamics and provides a means of studying fluid flow phenomena without the presence of rigid walls. Prior to the onset of a liquid bridge several important phenomena can be observed including advancing meniscus height (electrowetting), bulk fluid circulation (the Sumoto effect), and the ejection of charged droplets (electrospray). The interaction between surface, polarization, and displacement forces can be directly examined by varying applied voltage and bridge length. The electric field, assisted by gravity, stabilizes the liquid bridge against Rayleigh-Plateau instabilities. Construction of basic apparatus for both vertical and horizontal orientation along with operational examples, including thermographic images, for three liquids (e.g.
, water, DMSO, and glycerol) is presented.
Physics, Issue 91, floating water bridge, polar dielectric liquids, liquid bridge, electrohydrodynamics, thermography, dielectrophoresis, electrowetting, Sumoto effect, Armstrong effect
Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
Institutions: University of Exeter.
A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.
Biophysics, Issue 91, differential scanning fluorimetry, dissociation constant, protein-ligand interactions, StepOne, cooperativity, WcbI.
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
Cortical Source Analysis of High-Density EEG Recordings in Children
Institutions: UCL Institute of Child Health, University College London.
EEG is traditionally described as a neuroimaging technique with high temporal and low spatial resolution. Recent advances in biophysical modelling and signal processing make it possible to exploit information from other imaging modalities like structural MRI that provide high spatial resolution to overcome this constraint1
. This is especially useful for investigations that require high resolution in the temporal as well as spatial domain. In addition, due to the easy application and low cost of EEG recordings, EEG is often the method of choice when working with populations, such as young children, that do not tolerate functional MRI scans well. However, in order to investigate which neural substrates are involved, anatomical information from structural MRI is still needed. Most EEG analysis packages work with standard head models that are based on adult anatomy. The accuracy of these models when used for children is limited2
, because the composition and spatial configuration of head tissues changes dramatically over development3
In the present paper, we provide an overview of our recent work in utilizing head models based on individual structural MRI scans or age specific head models to reconstruct the cortical generators of high density EEG. This article describes how EEG recordings are acquired, processed, and analyzed with pediatric populations at the London Baby Lab, including laboratory setup, task design, EEG preprocessing, MRI processing, and EEG channel level and source analysis.
Behavior, Issue 88, EEG, electroencephalogram, development, source analysis, pediatric, minimum-norm estimation, cognitive neuroscience, event-related potentials
Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis
Institutions: University of Northern Colorado, Arizona State University, Iowa State University.
The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee prosthesis, and 2) contrast the effects of the proposed technique and that of using intact limb inertial properties on joint kinetic estimates during walking in unilateral, transtibial amputees. An oscillation and reaction board system was validated and shown to be reliable when measuring inertial properties of known geometrical solids. When direct measurements of inertial properties of the prosthesis were used in inverse dynamics modeling of the lower extremity compared with inertial estimates based on an intact shank and foot, joint kinetics at the hip and knee were significantly lower during the swing phase of walking. Differences in joint kinetics during stance, however, were smaller than those observed during swing. Therefore, researchers focusing on the swing phase of walking should consider the impact of prosthesis inertia property estimates on study outcomes. For stance, either one of the two inertial models investigated in our study would likely lead to similar outcomes with an inverse dynamics assessment.
Bioengineering, Issue 87, prosthesis inertia, amputee locomotion, below-knee prosthesis, transtibial amputee
Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS
Institutions: Massachusetts Institute of Technology.
Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae
, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.
Microbiology, Issue 77, Cellular Biology, Molecular Biology, Genetics, Biophysics, Saccharomyces cerevisiae, Microscopy, Fluorescence, Cell Biology, microscopy/fluorescence and time-lapse, budding yeast, gene expression dynamics, segmentation, lineage tracking, image tracking, software, yeast, cells, imaging
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers
Institutions: University of Michigan , University of Michigan .
Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2
, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4
. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6,
(see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8
. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.
Bioengineering, Issue 56, Genetics, DNA stretching, DNA, Axial Optical Tweezers, Single-Molecule Biophysics, Biophysics
A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology
Institutions: University of Ottawa, University of Ottawa, University of Calgary, University of Ottawa, University of Ottawa.
Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.
Bioengineering, Issue 88, cell stretching, tissue mechanics, nuclear mechanics, uniaxial, biaxial, anisotropic, mechanobiology
Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins
Institutions: University of Bayreuth.
Based on the natural sequence of Araneus diadematus
Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly α-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable β-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.
Bioengineering, Issue 75, Biochemistry, Chemistry, Materials Science, Molecular Biology, Cellular Biology, Proteins, Nanotechnology, materials (general), materials handling, nanodevices (mechanical), structural analysis, spider silk, electrospinning, microfibers, nonwoven, filter, mesh, biomaterials

domain: biology


Pavarotti To Perform For Angola Refugees
Luciano Pavarotti will stage a concert next month to raise money for Angolan child refugees living in Zambia, the United Nations (U.N.) refugee agency confirms. The Italian tenor wanted to remind theLuciano Pavarotti will stage a concert next month to raise money for Angolan child refugees living in Zambia, the United Nations (U.N.) refugee agency confirms. The Italian tenor wanted to remind the world of the plight of people affected by one of Africa's longest-running civil wars, which largely had been forgotten by the international community, the U.N. High Commissioner for Refugees said in a statement issued in the Zambian capital Lusaka.
Money raised at the May 28 concert, to take place in the Italian city of Modena, mainly would go toward health and education projects, the agency said. About 30 Angolan refugee children are to be chosen to perform at the concert.
Pavarotti, who is a U.N. Messenger of Peace, already has raised millions of dollars for the world's refugees. A concert in Modena last year, where Pavarotti topped the bill, raised $1.5 million for Afghan refugee children in Pakistan, while a 1999 concert collected $1 million for Kosovo refugees.
Angola's civil war began after the southwest African country gained independence from Portugal in 1975. It has claimed at least 500,000 lives and driven 4 million people -- about a third of the population -- from their homes.
Earlier this month the government and the UNITA rebel group signed a cease-fire agreement, the fourth since the war began, raising cautious optimism that the war may finally be over. Zambia hosts about 260,000 refugees, mainly from Angola and Congo.
Copyright 2002 Associated Press. All rights reserved. This material may not be published, broadcast, rewritten, or redistributed.


text:
drug testing issue hearings upcoming
The Willcox School
District (Arizona) is holding two public meetings
this week to gather ideas, comments and suggestions
regarding a proposed student drug testing policy.
meetings will be Tuesday, May 30, at 6:30 p.m., at
the Community Center, and Wednesday, May 31, at
noon, also at the Community Center.
The meetings will be for the same purpose, but two
different times are planned so that anyone wishing
to attend may choose a time that suits them, said
Sally White, director of Willcox Against Substance
Abuse. Everyone is invited. Following the two town
hall meetings and evidence of need, a policy would
be drafted and presented to the board in June or
July for approval, amendments or rejection.
"Illegal use of drugs is an increasing problem in
our society," said Superintendent Dr. Don Roberts.
"In my opinion, the district needs to send a strong
message. We want to give kids a way not to do (meth/illegal
drugs). If we can keep just one kid from trying (meth),
it's worth doing."
Roberts said that the district can verify a large
amount of drug/alcohol abuse, and therefore can
justify the random drug testing.
"We owe it to our community, parents and students.
This gives kids a chance to say no to peer pressure.
They can just say they don't want to get caught," he
Any seventh- through 12th-grade student
participating in extra-curricular activities,
including sports, fine arts, or others such as FFA
and FBLA, would be subject to random testing.
White said, "From the comments we have heard, it's
running about 7 to 1 for people who are supporting
student drug testing, but the ones who are not for
it are strongly against it."
She added that those against it seem to be against
it for one of two reasons. The first is that the
policy may infringe upon the rights of students or
they feel it is harassing students.
The second reason is that no punishment, such being
arrested is allowed for students who test positive
"We may not suspend, expel or academically punish a
child and we may not give results to police," White
said. "There are lots of consequences we can work
with, but those are against the law."
"We are working very hard on coming up with
someplace to refer students who test positive. The
cost would be deferred to the family, but we need to
work on a seamless transition so the procedure is
not so difficult that they won't do it," she said.
"All over the state we're grappling with that. All
over the nation, there are not enough resources to
help people in this situation, but in Arizona, we're
on the low end of the scale. Especially with kids.
"In order to make this (student drug-testing policy)
worthwhile, we need to be able to give students
testing positive and their families some course of
action to help them, such as counseling," White
Discussion so far has been for a plan to randomly
test students in extra-curricular activities. Those
testing positive could be removed from their
extra-curricular activity for some pre-determined
amount of time, whether it be a specified number of
games/practices/events or until they test negative.
"We're still in the information gathering phase. We
need to determine if it is time to do this in
Willcox or not. And if it is, we want to be able to
get it in place fairly quickly," she said.


text:
مجلة البحوث الاقتصادية والمالية
Volume 3, Numéro 1, Pages 7-23
This article is a study on the impact of the financial liberalization policy on economic growth in Algeria, made on the basis of an empirical analysis of annual time series to determine the effects of this policy on rates economic growth achieved during the period between 1980 and 2013. This led us to conclude that there is no causal relationship between financial liberalization and economic growth rates achieved in Algeria during this period. This explains the weakness of the banking liberalization and opening of the capital account in Algeria, despite the implementation of a series of financial reforms, banking and financial liberalization reforms in 1990 since the release of the law No. 90-10 on money and credit. These reforms have not yielded acceptable results in improving the role of the Algerian banking and financial institutions in the growth and operation of financial resources. The improved financial and monetary indicators and economic growth rates achieved in the last two decades by Algeria, mainly due to other factors such as the rising price of oil and the volume of foreign exchange reserves.
Algeria, economic growth, financial liberalization, law No. 90-10-
بوسالم أبو بكر
بن علال بلقاسم

domain: literature