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Archive for the ‘Image Processing/Computing’ Category


Imaging  Living Cells and Devices, Presentation by Danut Dragoi, PhD, LPBI group

Imaging living cells is for a good number of years a hot place in Biology, Physics, Chemistry as well as Engineering and Technology for producing specific devices to visualize living cells. In this presentation is shown my opinion on this topic regarding actual status of applied technology for visualizing living cells as well as small small areas of interest.

Slide #1

Slide1

Slide #2

Slide2

As an overview, slide #2 describes: higher resolution imaging of living cells based on advanced CT and MicroCT scanners, and their actual technological trend,  advanced optical microscopy, optical magnetic imaging of living cells, and conclusion.

Slide #3

Slide3

Slide #3 describes a schematic of a computing tomography applied to a single cell, see the inside URL address. The work is in progress as a SBIR application of a group of researchers from Arizona State.The partial section of the cell is supposed to  reveal the contents of the cell, which is very important in Biology and Medicine.

Slide #4

Slide4

Slide #4 describes the principle of computed tomography for relative small objects that are expose by a soft x-ray source on the left, an x-ray detector screen that takes the x-ray projection radiography for the sample on the right. The sample is rotated discretely a small number of degrees and pictures recorded. Depending on the absorption of the sample, the reconstructed 3D object is possible. The resolution of the reconstructed object is a function of the number of pixels as well as the pitch distance d (in the slide 0.127 microns). Because the sample is rotated, the precision of the axis of rotation is very important and becomes a challenging task for small objects.

Slide #5

Slide5

Slide #5 shows a sample taken from the URL address given below the picture. It represents an insect and the future CT development is expected to produce similar images for mono living cells.

Slide #6

Slide6

For many Bio-labs the reverse optical microscope is the working horse. The slide above shows a such microscope with a culture cell inside a transparent box. The picture can be found at the address shown inside the slide.

Slide #7

Slide7

Slide #7 describes an innovative digital microscope from Keyence in which we can observe any object entirely in focus, a 20x greater depth-of-field than an optical microscope, we can view objects from any angle, and measure lengths directly on screen.

Slide #8

Slide8

Slide #8 shows an actual innovative digital microscope from Keyence, see the website address at the bottom of the slide.

Slide #9

Slide9

As we know, the samples visualized by a common optical microscope have to be flat on the surface to be visualized because there is no clear image above and below the focal plane, which is the surface of the sample. For a con-focal microscope the situation is changed. Objects can be visualized at different depths and image files recorded can reconstruct as 3D image object.

Slide #10

Slide10

Slide #10 describes the principle of a con-focal microscope, in which a green laser on left side excites molecules of the specimen at a given depth of focusing, the molecules emit on red light (less energetic than green light) that go all way to the photo-multiplier, which has a small pinhole aperture in front of it that limits the entrance of red rays (parasitic light) from out of range area. More details can be found at the URL address given at the bottom of the slide.

Slide #11

Confocal microscopy Leica

Slide #11 shows a sample of a living specimen taken with a Leica micro-system, see the website address inside the slide.

Slide #12

Slide12

Slide #12 shows the principle of fluorescent microscope and how it works. A light source is filtered to allow blue light (energetic photons for excitation of the molecules of the specimen), the green light emitted is going through objective and ocular lenses and further to the photo-multiplier or digital camera.

Slide #13

Slide13

For their discovery of fluorescent microscopy, Eric Betzig, William Moerner and Stefan Hell won the Nobel Prize in Chemistry on 2014,  for “the development of super-resolved fluorescence microscopy,” which brings optical microscopy into the nano-dimension.

Slide #14

Slide14

Slide #14 introduces the improvement on micro CT scanners for imaging living cells which now is on R & D under heavy development.  The goal is to visualize the interior of living cells. Challenging tasks are: miniaturization, respond to customer needs, low cost, and versatility.

Slide #15

Slide15

Slide #15 shows the schematic for an optical magnetic imaging microscope for visualizing living cells with one dimension less than 500 nm. The website address gven describes in details the working principle.

Slide #16

Slide16

Slide #16 shows the picture of a hand held microscope that is useful on finding spot cancer in moments, ses the website.

Slide #17

Slide17

Slide #17 shows a hand held MRI that connects to an iPhone. It is useful device for detecting cancer cells.

Slide #18

Slide18

Slide #18 shows in comparison a portable NMR device, left side, and a Lab NMR instrument whose height is greater than 5 Ft. The spectrum in the left side is that of Toluene and a capillary sample holder is shown also next to the magnetic device.

Slide #19

Slide19

Slide #19 shows that the hand held MRI can recognize complex molecules,  can diagnose cancer faster, can be connected to a smartphone, and be accurate on precise measurements.

Slide #20

Slide20

An optical dental camera is shown in slide #20. It is less then $100 and a USB cable can connect to a computer. It is very useful for every family in checking the status of the teeth and gums.

Slide #21

Slide21

For detecting dental cavities the x-ray source packaged as a camera and the sensor that connects to a computer are very useful tools in a dental office.

Slide #22

Slide22

Slide #22 shows the conclusions of the presentation, in which we summarize: the automated con-focal microscope partially satisfies the actual needs for imaging of living cells, the optical magnetic imaging microscope for living cells is a promising technique,
a higher resolution is needed on all actual microscopes,  the advanced CT and Micro CT scanners provide a new avenue on the investigation of living cells, more research needed
on hand-held MRI, which is a new solution for complex molecules recognition including cancer

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3D Imaging of Cancer Cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

3D Imaging of Cancer Cells Could Lead to Improved Ability of Pathologists and Radiologists to Plan Cancer Treatments and Monitor Cell Interactions

Dark Daily Apr 8th 2016        Jon Stone

https://www.linkedin.com/pulse/3d-imaging-cancer-cells-could-lead-improved-ability-plan-joseph-colao

 

3D Imaging of Cancer Cells Could Lead to Improved Ability of Pathologists and Radiologists to Plan Cancer Treatments and Monitor Cell Interactions.

New technology from researchers at the University of Texas Southwestern Medical Center enables the ability to study cancer cells in their native microenvironments.

Imaging research is one step closer to giving clinicians a way to do high-resolution scans of malignant cells in order to diagnose cancer and help identify useful therapies. If this technology were to prove successful in clinical studies, it might change how anatomic pathologists and radiologists diagnose and treat cancer.

Researchers at the University of Texas Southwestern Medical Center developed a way to create near-isotropic, high-resolution scans of cells within their microenvironments. The process involves utilizing a combination of two-photonBessel beams and specialized filtering.

New Imaging Approach Could be Useful to Both Pathologists and Radiologists

In a recent press release, senior author Reto Fiolka, PhD, said “there is clear evidence that the environment strongly affects cellular behavior—thus, the value of cell culture experiments on glass must at least be questioned. Our microscope is one tool that may bring us a deeper understanding of the molecular mechanisms that drive cancer cell behavior, since it enables high-resolution imaging in more realistic tumor.”

In a study in Developmental Cell, Erik S. Welf, PhD, et al, described the new microenvironmental selective plane illumination microscopy (meSPIM). When developing the technology, the team outlined three goals:

1. The microscope design must not prohibitively constrain microenvironmental properties.

2. Spatial and temporal resolution must match the cellular features of interest.

3. Spatial resolution must be isotropic to avoid spatial bias in quantitative measurements.

This new technology offers pathologists and medical laboratory scientists a new look at cancer cells and other diseases. The study notes that meSPIM eliminates the influence of stiff barriers, such as glass slide covers, while also allowing a level of control over both mechanical and chemical influences that was previously impossible.

Early meSPIM Research Reveals New Cell Behaviors

Early use of meSPIM in observing melanoma cells is already offering new insights into the relationship between the cell behavior of cellular- and subcellular-scale mechanisms and the microenvironment in which these cells exist. The study notes, “The ability to image fine cellular details in controllable microenvironments revealed morphodynamic features not commonly observed in the narrow range of mechanical environments usually studied in vitro.”

One such difference is the appearance of blebbing. Created by melanoma cells and lines, these small protrusions are thought to aid in cell mobility and survival. Using meSPIM, observers could follow the blebbing process in real-time. Formation of blebs on slides and within an extracellular matrix (ECM) showed significant differences in both formation and manipulation of the surrounding microenvironment.

The team is also using meSPIM to take a look at membrane-associated biosensor and cytosolic biosensor signals in 3D. They hope that investigation of proteins such as phosphatidylinositol 3-kinase (PI3K) and protein kinase C will help to further clarify the roles these signals play in reorientation of fibroblasts.

meSPIM combined with computer vision enables imaging, visualization, and quantification of how cells alter collagen fibers over large distances within an image volume measuring 100 mm on each side. (Photo Copyright: Welf and Driscoll et al.)

The research team believes this opens new possibilities for studying diseases at a subcellular level, saying, “Cell biology is necessarily restricted to studying what we can measure. Accordingly, while the last hundred years have yielded incredible insight into cellular processes, unfortunately most of these studies have involved cells plated onto flat, stiff surfaces that are drastically different from the in vivo microenvironment …

“Here, we introduce an imaging platform that enables detailed subcellular observations without compromising microenvironmental control and thus should open a window for addressing these fundamental questions of cell biology.”

Limitations of meSPIM

One significant issue associated with the use of meSPIM is the need to process the large quantity of data into useful information. Algorithms currently allow for automatic bleb detection. However, manual marking, while time consuming, still provides increased accuracy. Researchers believe the next step in improving the quality of meSPIM scans lie in computer platforms designed to extract and process the scan data.

Until this process is automated, user bias, sample mounting, and data handling will remain risks for introducing errors into the collected data. Yet, even in its early stages, meSPIM offers new options for assessing the state of cancer cells and may eventually provide pathologists and radiologists with additional information when creating treatment plans or assessments.

 

Seeing cancer cells in 3-D (w/ Video)

http://phys.org/news/2016-02-cancer-cells-d-video.html

 

Cancer in 3-D

http://cdn.phys.org/newman/csz/news/800/2016/cancerin3d.png

Extracted surfaces of two cancer cells. (Left) A lung cancer cell colored by actin intensity near the cell surface. Actin is a structural molecule that is integral to cell movement. (Right) A melanoma cell colored by PI3-kinase activity near the cell surface. PI3K is a signaling molecule that is key to many cell processes. Credit: Welf and Driscoll et al.

Cancer cells don’t live on glass slides, yet the vast majority of images related to cancer biology come from the cells being photographed on flat, two-dimensional surfaces—images that are sometimes used to make conclusions about the behaviour of cells that normally reside in a more complex environment. But a new high-resolution microscope, presented February 22 in Developmental Cell, now makes it possible to visualize cancer cells in 3D and record how they are signaling to other parts of their environment, revealing previously unappreciated biology of how cancer cells survive and disperse within living things.

“There is clear evidence that the environment strongly affects cellular behavior—thus, the value of cell culture experiments on glass must at least be questioned,” says senior author Reto Fiolka, an optical scientist at the University of Texas Southwestern Medical Center. “Our is one tool that may bring us a deeper understanding of the molecular mechanisms that drive cancer cell behavior, since it enables high-resolution imaging in more realistic tumor environments.”

Read more at: http://phys.org/news/2016-02-cancer-cells-d-video.html#jCp

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Nanophotonics Applications

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Copper Plasmonics Explored for Nanophotonics Applications

http://www.photonics.com/Article.aspx?AID=58484

MOSCOW, March 22, 2016 — Experimental demonstration of copper components has expanded the list of potential materials suited to nanophotonic devices beyond gold and silver.

According to researchers from the Moscow Institute of Physics and Technology (MIPT), copper components are not only just as good as components based on noble metals, such as gold and silver, they can be easily implemented in integrated circuits using industry-standard fabrication processes. Gold and silver, as noble metals, may not enter into the requisite chemical reactions to create nanostructures readily and require expensive, difficult processing steps.

Nanoscale copper plasmonic waveguides on a silicon chip in a scanning near-field optical microscope (left) and their image obtained using electron microscopy (right).

Nanoscale copper plasmonic waveguides on a silicon chip in a scanning near-field optical microscope (left) and their image obtained using electron microscopy (right). Courtesy of MIPT.

In nanophotonics, the diffraction limit of light is overcome by using metal-dielectric structures. Light may be converted into surface plasmon polaritons, surface waves propagating along the surface of a metal, which make it possible to switch from conventional 3D photonics to 2D surface plasmon photonics, also known as plasmonics. This allows control of light at the 100-nm scale, far beyond the diffraction limit.

Now researchers from MIPT’s Laboratory of Nanooptics and Plasmonics have found a solution to the problems posed by noble metals. Based on a generalization of the theory for so-called plasmonic metals, in 2012 they found that copper as an optical material is not only able to compete with gold, but it can also be a better alternative. Unlike gold, copper can be easily structured using wet or dry etching. This gives a possibility to make nanoscale components that are easily integrated into silicon photonic or electronic integrated circuits.

Silicon chip with nanoscale copper plasmonic components.

Silicon chip with nanoscale copper plasmonic components. Courtesy of MIPT.

It took more than two years for the researchers to purchase the required equipment, develop the fabrication process, produce samples, conduct several independent measurements and confirm their hypothesis experimentally.

“As a result, we succeeded in fabricating copper chips with optical properties that are in no way inferior to gold-based chips,” says the research leader Dmitry Fedyanin. “Furthermore, we managed to do this in a fabrication process compatible with the CMOS technology, which is the basis for all modern integrated circuits, including microprocessors. It’s a kind of revolution in nanophotonics.”

The researchers said that the optical properties of thin polycrystalline copper films were determined by their internal structure, and that controlling this structure to achieve and consistently reproduce the required parameters in technological cycles was the most difficult task.

Having demonstrated copper’s suitable material characteristics, as well as nanoscale manufacturing capability, the researchers believe the devices could be integrated with both silicon nanoelectronics and silicon nanophotonics. Such technologies could enable LEDs, nanolasers, highly sensitive sensors and transducers for mobile devices, and high-performance optoelectronic processors with several tens of thousands of cores for graphics cards, personal computers and supercomputers.

“We conducted ellipsometry of the copper films and then confirmed these results using near-field scanning optical microscopy of the nanostructures. This proves that the properties of copper are not impaired during the whole process of manufacturing nanoscale plasmonic components,” says Dmitry Fedyanin.

The research was published in Nano Letters (doi: 10.1021/acs.nanolett.5b03942).

 

Ultralow-Loss CMOS Copper Plasmonic Waveguides

Surface plasmon polaritons can give a unique opportunity to manipulate light at a scale well below the diffraction limit reducing the size of optical components down to that of nanoelectronic circuits. At the same time, plasmonics is mostly based on noble metals, which are not compatible with microelectronics manufacturing technologies. This prevents plasmonic components from integration with both silicon photonics and silicon microelectronics. Here, we demonstrate ultralow-loss copper plasmonic waveguides fabricated in a simple complementary metal-oxide semiconductor (CMOS) compatible process, which can outperform gold plasmonic waveguides simultaneously providing long (>40 μm) propagation length and deep subwavelength (∼λ2/50, where λ is the free-space wavelength) mode confinement in the telecommunication spectral range. These results create the backbone for the development of a CMOS plasmonic platform and its integration in future electronic chips.

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Chromatography and Mass Spectroscopy

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Optimization of Chromatography in the Lab

Sanji Bhal & Karim Kassam; ACD/Labs

While analytical laboratories may still rely to some extent on trial-and-error approaches, there is agreement that this is increasingly less effective as systems become more complex. Regulatory bodies are putting increasing pressure on pharmaceutical companies to incorporate Quality by Design (QbD) approaches throughout the drug development process. QbD is defined in the ICH Q8 guideline as “A systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management.”

Developing effective and robust separations methods can be a very time-consuming process. A comprehensive approach to method development would be thorough investigation of the design space for any given mixture or sample including buffer, column, solvent, time, temperature, etc. Given the time constraints and limited resources in any R&D laboratory, however, this type of broad scope investigation is unrealistic.

Modeling for the optimization of chromatographic separations of small molecules has been successfully used for approximately 30 years. A large number of articles have been published on this topic by L. Snyder, P. Janderra, P. Schoenmakers et al. Modeling of chromatographic separations continues to be of interest because as the science of chromatographic separations continues to evolve, modeling techniques must evolve with them to support the needs of the community.

New types of chromatographic techniques (UHPLC, HILIC, ion exchange chromatography, etc.) have demanded the need for new modeling tools. This also led to the need for translation of methods from one technique to newer techniques (HPLC to UHPLC, for example). Furthermore, as pharmaceutical R&D has expanded investigation of new drugs from small molecules to proteins and bio-molecules, many of the old rules no longer apply.

The ability to model the behavior of a sample in silico provides chromatographers with a number of advantages:

Greater efficiency in method development—it is difficult to estimate the number of hours required to identify a suitable method for separation of a mixture. An experienced scientist will rely on their knowledge while an inexperienced colleague may struggle with the same separation. As the number of experienced chromatographers decreases across organizations, and the existing scientists are retiring, software to assist those less experienced becomes more attractive.

With such a large number of variables (temperature, gradient, pH, salt concentration, etc.) it is advantageous to use all available knowledge and tools to ‘get ahead’. Increased efficiency can be realized not only in identifying an optimal method faster but also increasing throughput and decreasing scale-up time.

Risk mitigation through robust methods—this is the ideal result of a method development project. By applying QbD principles and understanding the analytical design space of a sample, the chromatographer can understand, reduce, and control sources of variability; and use this information to create a method that is reliable and robust. Simulation of methods provides scientists the luxury of thoroughly investigating method development space with limited consumption of resources and time, for the best result.

Economic considerations—while there is a cost in man hours and time spent on method development, there is also unrecoverable expenditure on consumables (solvents, columns, etc.). In being able to investigate chromatographic space in silico, this time and expenditure can be greatly reduced.

Green chemistry—the ability to model separations not only reduces the volume of waste, it may also help us reduce environmental impact. Consider the case of acetonitrile shortages in recent years. The ability to use alternative methods, i.e., replacing acetonitrile with methanol, not only lead to reduced cost but also has the side effect of more environmentally safe waste.

Software provided with chromatographic instruments delivers many useful capabilities to execute experimental runs and control instruments. Simulation software, however, is typically purchased separately. Several commercial software packages are available, i.e., DryLab, ACD/LC Simulator (from ACD/Labs), ChromSword, and Osiris, each of which provides different advantages and limitations (an exhaustive list is outside the scope of this article).

Commercially available method optimization software is typically built on one of three models—simulations based on molecular structure, retention based modeling, and statistical modeling. Each has its pros and cons with details in their implementation that appeal to different applications.

Data input—flexibility of data import into a system from the instrument is an important consideration when dealing with multiple experiments under varying conditions. Lack of standardization of chromatographic data formats today, however, means that unless data from separations is transferred into Excel or similar software, scientists are left to transcribe information from one system to another. Direct data import from chromatographic runs into third-party modeling software, in the instrument format, is ideal since it avoids transcription errors and saves time in data input. ACD/Labs provides the only software (ACD/LC Simulator and ACD/GC Simulator) with instrument vendor-agnostic support of analytical data at this time.

Data visualization—the ability to review and interrogate data is of utmost importance in method development and optimization, and software vendors implement various tools to meet chromatographers’ requirements. While 3D modeling, offered by DryLab, has enjoyed popularity in the community, the question of applicability still remains. A significant amount of data input (upwards of 45 injections is not unreasonable for simultaneous optimization of 3 factors) is required for effective 3D modeling, which in itself is counter-intuitive if time and resource efficiency is the ultimate goal.

Automation—ACD/AutoChrom (from ACD/Labs) and ChromSword both provide automation through instrument control. AutoChrom provides automation of the most popular Waters Empower and Agilent ChemStation systems and keeps the scientist in control by allowing user input at key stages of the method development process. This software is best suited for challenging separations such as stability indicating methods and forced degradation studies.

Custom Modeling—while third-party modeling software may cover a broad range of structure and method development space, there is nothing better than the ability for scientists to create their own models. ACD/LC Simulator was the only software known to the authors at the time of publication that offers this capability. Work published by world class chromatographers Patrik Pettersson and Mel Eureby demonstrates the use of ACD/LC Simulator in successfully modelling protein and HILIC separations.

Reverse phase HPLC, temperature/gradient optimization as modeled in ACD/LC Simulator. (Credit:  ACD/Labs )

Reverse phase HPLC, temperature/gradient optimization as modeled in ACD/LC Simulator. (Credit: ACD/Labs )

Physicochemical property predictions such as logD and pKa can also help in method development and optimization. In a general sense, being able to predict behavior with respect to pH can offer insights into method development challenges. ChromSword and ACD/Labs software both provide property predictions, and the latter have been leaders in this field for almost two decades with applications across various areas of research and industries.

As the science of separations evolves and the compounds of interest change, the software to support scientific research and development will need to develop alongside. Software vendors need to satisfy the needs of their customer organizations in releasing the time of valuable scientists for innovation thus releasing them from monotonous and tedious tasks. If your organization has yet to invest in software for modeling separations, it will likely come in the future and many of the topics raised here should be kept in mind to ensure you get the best return on investment.

 

Tissue Imaging Mass Spec Detects Early Lipid Changes in Acute Kidney Injury

University of Alabama at Birmingham researchers have made a microscopic snapshot of the early renal lipid changes in acute kidney injury, using a laser-scanning method called MALDI tissue imaging to localize the changes.
These disease-model results, recently published in American Journal of Physiology’s Renal Physiology, show an example of the power of MALDI tissue imaging. MALDI tissue imaging is now available at UAB, and it will be able to aid basic and clinical biomedical research across the campus, said corresponding author Janusz Kabarowski, Ph.D., associate professor of microbiology.
“I think the opportunity to integrate this into existing UAB research centers to facilitate grants is immense,” Kabarowski said. “It can be utilized for any tissue damage. For drugs that can be imaged with MALDI imaging mass spectrometry, you can tell where in a slice of tissue the drugs get to, with obvious implications for testing candidate therapeutic agents in cancer research too. We can capture—at the molecular level—a moment in time.”
The imaging has the power to reveal spatial distribution of complex biochemical processes in an organism, showing where changes in proteins or small molecules take place. Unlike chemical stains, immunohistochemical tags or radioactive labels, it does not require a priori knowledge of the target compounds.
Acute kidney injury is a leading cause of hospital illness or death in critically ill patients. In a mouse model of the injury used by Kabarowski and colleagues, kidneys were made ischemic for 30 minutes. Six hours after reperfusion, and before gross kidney damage was seen, the kidneys were removed and cut in half. The lipids were extracted from one of the halves; the other was flash frozen and cut into thin sections that were mounted on specially coated slides.
Extracted lipids were analyzed using SWATH mass spectrometry, and the UAB researchers found that four were significantly changed at six hours (all were increases). Three of the lipids were ether-linked phospholipids, including a plasmalogen, a type of ether phospholipid thought to have protective anti-oxidant properties. They also found that the levels of these ether-linked phospholipids correlated with levels of plasma creatinine, a marker of acute kidney injury. This suggests a causal or a protective role for them in acute kidney injury, and also suggests they may be an effective early biomarker for injury.
The researchers then used MALDI tissue imaging to find where the most abundant of the ether-linked phospholipids was concentrated. In MALDI, a powerful laser scans the thin tissue section after application of a matrix material by vacuum sublimation, knocking the lipid ions off from the surface of the tissue. The MALDI time-of-flight mass spectrometry and ion fragmentation then allowed identification of the proximal tubules of the kidney as the place where the ether-linked phospholipids were concentrated. The proximal tubules are known to be most prone to developing ischemia-related injury.
Besides Kabarowski, authors of “Early lipid changes in acute kidney injury using SWATH lipidomics coupled with MALDI tissue imaging” are co-first authors Sangeetha Rao, M.D., fellow in the UAB Pediatric Critical Care Medicine, and Kelly B. Walters, UAB departments of Chemistry and Microbiology; Landon Wilson and Stephen Barnes, Ph.D., UAB Department of Pharmacology and Toxicology, Targeted Metabolomics and Proteomics Laboratory; Bo Chen, Ph.D., Subhashini Bolisetty, Ph.D., and Anupam Agarwal, M.D., UAB Division of Nephrology and the Nephrology Research and Training Center; and David Graves, UAB Department of Chemistry.
MALDI imaging mass spectrometry stands for “matrix-assisted laser desorption ionization” imaging mass spectrometry. SWATH mass spectrometry stands for “sequential window acquisition of all theoretical spectra” mass spectrometry.

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Laser Therapy Opens Blood-Brain Barrier

Curator: Larry H. Bernstein, MD, FCAP

 

Laser Surgery Opens Blood-Brain Barrier to Chemotherapy

http://www.photonics.com/Article.aspx?AID=58445

ST. LOUIS, March 11, 2016 — A laser probe has been used to open the brain’s protective cover, enabling delivery of chemotherapy drugs to patients with glioblastoma — the most common and aggressive form of brain cancer.

In a pilot study conducted by the Washington University School of Medicine in St. Louis, Mo., 14 patients with glioblastoma underwent minimally invasive laser surgery to treat a recurrence of their tumors. Heat from the laser was already known to kill brain tumor cells but, unexpectedly, the researchers found that the technology penetrated the blood-brain barrier.

“The laser treatment kept the blood-brain barrier open for four to six weeks, providing us with a therapeutic window of opportunity to deliver chemotherapy drugs to the patients,” said neurosurgery professor Eric Leuthardt, MD, who also treats patients at Barnes-Jewish Hospital. “This is crucial because most chemotherapy drugs can’t get past the protective barrier, greatly limiting treatment options for patients with brain tumors.

The team is still closely following the patients, though early results indicate they are doing better on average, in terms of survival and clinical outcomes, than what the researchers would expect with other treatment methods.

Glioblastomas are one of the most difficult cancers to treat. Most patients diagnosed with this type of brain tumor survive just 15 months, according to the American Cancer Society.

The research is part of a larger phase II clinical trial that will involve 40 patients. Twenty patients were enrolled in the pilot study, 14 of whom were found to be suitable candidates for the minimally invasive laser surgery, a technology that Leuthardt helped pioneer.

The laser technology was approved by the FDA in 2009 as a surgical tool to treat brain tumors. The Washington team’s research marks the first time the laser has been shown to disrupt the blood-brain barrier, which shields the brain from harmful toxins but inadvertently blocks potentially helpful drugs, such as chemotherapy.

As part of the trial, doxorubicin, a widely used chemotherapy, was delivered intravenously to 13 patients in the weeks following the laser surgery. Preliminary data indicate that 12 patients showed no evidence of tumor progression during the short, 10-week time frame of the study. One patient experienced tumor growth before chemotherapy was delivered; the tumor in another patient progressed after chemotherapy was administered, the researcher reported.

The laser surgery was well-tolerated by the patients in the trial; most went home one to two days afterward, and none experienced severe complications. The surgery was performed while a patient lies in an MRI scanner, providing the neurosurgical team with a real-time look at the tumor. Using an incision of only 3 mm, a neurosurgeon robotically inserted the laser to heat up and kill brain tumor cells at a temperature of about 150 °F.

“The laser kills tumor cells, which we anticipated,” said Leuthardt. “But, surprisingly, while reviewing MRI scans of our patients, we noticed changes near the former tumor site that looked consistent with the breakdown of the blood-brain barrier.”

Leuthardt confirmed and further studied these imaging findings with study co-author Dr. Joshua Shimony, a professor of radiology at Washington University.

The researchers, including co-corresponding author Dr. David Tran, a neuro-oncologist now at the University of Florida, performed follow-up testing, which showed that the degree of permeability through the blood-brain barrier peaked one to two weeks after surgery but that the barrier remained open for up to six weeks.

Other successful attempts to breach the barrier have left it open for only a short time — about 24 hours — not long enough for chemotherapy to be consistently delivered, or have resulted in only modest benefits, the researchers said. The laser technology leaves the barrier open for weeks — long enough for patients to receive multiple treatments with chemotherapy. Further, the laser only opens the barrier near the tumor, leaving the protective cover in place in other areas of the brain. This has the potential to limit the harmful effects of chemotherapy drugs in other areas of the brain, the researchers said.

The findings also suggest that other approaches, such as cancer immunotherapy — which harnesses cells of the immune system to seek out and destroy cancer — could also be useful for patients with glioblastomas.

The researchers are planning another clinical trial that combines the laser technology with chemotherapy and immunotherapy, as well as trials to test targeted cancer drugs that normally can’t breach the blood-brain barrier.

The research was published in Plos One (doi: 10.1371/journal.pone.0148613).

 

Hyperthermic Laser Ablation of Recurrent Glioblastoma Leads to Temporary Disruption of the Peritumoral Blood Brain Barrier

Poor central nervous system penetration of cytotoxic drugs due to the blood brain barrier (BBB) is a major limiting factor in the treatment of brain tumors. Most recurrent glioblastomas (GBM) occur within the peritumoral region. In this study, we describe a hyperthemic method to induce temporary disruption of the peritumoral BBB that can potentially be used to enhance drug delivery.

 Methods

Twenty patients with probable recurrent GBM were enrolled in this study. Fourteen patients were evaluable. MRI-guided laser interstitial thermal therapy was applied to achieve both tumor cytoreduction and disruption of the peritumoral BBB. To determine the degree and timing of peritumoral BBB disruption, dynamic contrast-enhancement brain MRI was used to calculate the vascular transfer constant (Ktrans) in the peritumoral region as direct measures of BBB permeability before and after laser ablation. Serum levels of brain-specific enolase, also known as neuron-specific enolase, were also measured and used as an independent quantification of BBB disruption.

Results

In all 14 evaluable patients, Ktrans levels peaked immediately post laser ablation, followed by a gradual decline over the following 4 weeks. Serum BSE concentrations increased shortly after laser ablation and peaked in 1–3 weeks before decreasing to baseline by 6 weeks.

Conclusions   

The data from our pilot research support that disruption of the peritumoral BBB was induced by hyperthemia with the peak of high permeability occurring within 1–2 weeks after laser ablation and resolving by 4–6 weeks. This provides a therapeutic window of opportunity during which delivery of BBB-impermeant therapeutic agents may be enhanced.

Trial Registration  

ClinicalTrials.gov NCT01851733

Citation: Leuthardt EC, Duan C, Kim MJ, Campian JL, Kim AH, Miller-Thomas MM, et al. (2016) Hyperthermic Laser Ablation of Recurrent Glioblastoma Leads to Temporary Disruption of the Peritumoral Blood Brain Barrier. PLoS ONE 11(2): e0148613.  http://dx.doi.org:/10.1371/journal.pone.0148613

Glioblastoma (GBM) is the most common and lethal malignant brain tumor in adults [1]. Despite advanced treatment, median survival is less than 15 months, and fewer than 5% of patients survive past 5 years [2, 3]. Effective treatment options for recurrent GBM remain very limited and much of research and development efforts in recent years have focused on this area of greatly unmet needs. Up to 90% of recurrent tumors develop within the 2–3 cm margin of the primary site and are thought to arise from microscopic glioma cells that infiltrate the peritumoral brain region prior to resection of the primary tumor [4, 5]. Therefore elimination of infiltrative GBM cells in this region likely will improve long-term disease control.

Inadequate CNS delivery of therapeutic drugs due to the blood brain barrier (BBB) has been a major limiting factor in the treatment of brain tumors. The presence of contrast enhancement on standard brain MRI qualitatively reflects a disrupted state of the BBB. For this reason, drug access to the viable contrast enhanced tumor rim is likely significantly higher than to the peritumoral region, which usually does not have contrast enhancement [6, 7]. Evidence supporting this hypothesis came from studies in which drug levels of cytotoxic agents were sampled in tumors and the surrounding brain tissue at the time of surgery or autopsy. Drug concentrations were at the highest in the enhancing portion of tumors, and then rapidly decreased up to 40 fold lower by 2–3 cm distance from the viable tumor edge [810]. Overall, these observations suggest that the BBB and its integrity negatively correlate with delivery and potentially therapeutic effects of BBB impermeant drugs.

To circumvent the BBB problem in local drug delivery, recent approaches have focused on bypassing it. A previously described method is the use of Gliadel, a polymer wafer impregnated with the chemotherapeutic agent carmustine (BCNU) and placed intra-operatively in the resection cavity to bypass the BBB. This approach resulted in a statistically significant but modest survival advantage in both newly diagnosed and recurrent GBM [1113]. The modest benefit of Gliadel could be due to the short duration of drug delivery as nearly 80% of BCNU is released from the wafer over a period of only 5 days [14]. This observation further supports the notion that the BBB is critical to chemotherapy effect. However, Gliadel is not widely utilized as it requires an open craniotomy and can impair wound healing. Another approach of bypassing the BBB is the convection-enhanced delivery system in which a catheter is surgically inserted into the tumor to deliver chemotherapy [15]. This procedure requires prolonged hospitalization to maintain the external catheter to prevent serious complications and as a result has not been used extensively.

The role of hyperthermia in inducing BBB disruption has been previously described in animal models of CNS hyperthermia. In a rodent model of glioma, the global heating of the mouse’s head to 42°C for 30 minutes in a warm water bath significantly increased the brain concentration of a thermosensitive liposome encapsulated with adriamycin chemotherapy [16]. To effect more locoregional hyperthermia, retrograde infusion of a saline solution at 43°C into the left external carotid artery in the Wistar rat reversibly increased BBB permeability to Evans-blue albumin in the left cerebral hemisphere [17]. In another approach, neodymium-doped yttrium aluminum garnet (Nd:YAG) laser-induced thermotherapy to the left forebrain of Fischer rats resulted in loco-regional BBB disruption as evidenced by passage of Evans blue dye, serum proteins (e.g. fibrinogen & IgM), and the chemotherapeutic drug paclitaxel for up to several days after thermotherapy [18]. The effect of hyperthermia on the BBB of human brain has not been examined.

Here we describe an approach to induce sustained, local disruption of the peritumoral BBB using MRI-guided laser interstitial thermal therapy, or LITT. The biologic effects and correlation with MRI findings of LITT have been studied in both animal and human models since the development of LITT over twenty years ago. A well-described zonal distribution of histopathological changes with corresponding characteristic MR imaging findings centered on the light-guide track replace the lesion targeted for thermal therapy. The central treatment zone shows development of coagulative necrosis with complete loss of normal neurons or supporting structures immediately following therapy, corresponding to hyperintense T1-weighted signal intensity relative to normal brain [1922]. The peripheral zone of the post-treatment lesion is characterized by avid enhancement with intravenous gadolinium contrast agents, which peaks several days following thermal therapy and persists for many weeks after the procedure. Gadolinium contrast enhancement in the brain following LITT is due to leakage of gadolinium contrast into the extravascular space across a disrupted BBB [2023]. The perilesional zone of hyperintense signal intensity of FLAIR-weighted images develops within 1–3 days of thermal treatment and persists for 15–45 days [22].

We demonstrate that in addition to cytoreductive ablation of the main recurrent tumor, hyperthermic exposure of the peritumoral region resulted in localized, lasting disruption of the BBB as quantified by dynamic contrast-enhanced MRI (DCE-MRI) and serum levels of brain-specific enolase (BSE), thus providing a therapeutic window of opportunity for enhanced delivery of therapeutic agents.

Table 1. Patient Baseline Demographics and Characteristics.
TMZ/RT: Stupp protocol of 60 Gy radiotherapy plus concurrent 75mg/m2 daily temozolomide. Doxorubicin treatment: Timing of 20mg/m2 IV weekly doxobubicin treatment after LITT. Early = Starting within 1 week after LITT; Late = Starting at 6 weeks after LITT.  http://dx.doi.org:/10.1371/journal.pone.0148613.t001
……
Quantitative measurement of LITT-induced peritumoral BBB disruption by DCE-MRI

Brain MRI obtained within 48 hours following LITT showed the targeted tumor replaced by a post-treatment lesion corresponding to the volume of treated tissue on intraoperative thermometry maps. The post-treatment lesion lost the original rim of tumor-associated contrast enhancement and instead demonstrated central hyperintense T1-weighted signal compared to the pre-treated tumor and normal brain and a faint, newly developed discontinuous rim of peripheral contrast enhancement extending beyond the original tumor-associated enhancing rim (Fig 2A). These findings are consistent with a loss of viable tumor tissue caused by LITT, thus achieving an effective cytoreduction similar to open surgical resection. Of note, the rim of new peripheral contrast enhancement persisted for at least the next 28 days (Fig 2B–2E). Perilesional edema qualitatively evaluated on FLAIR-weighted images increased from pretreatment imaging at week 2 and persisted at week 4 following LITT (Fig 2F–2I). Perilesional edema decreased on subsequent MRI examinations. These findings qualitatively indicate that peritumoral BBB is disrupted by LITT and that the disruption peaks within approximately 2 weeks after the procedure.

……

Fig 3 demonstrates the Ktrans time curves for our cohort of patients. In all subjects the Ktrans in the ROIs within the enhancing ring around the ablated tumor is highly elevated in the first few days after the procedure and then progressively decreases at approximately the 4-week time point. The bottom right subplot in Fig 3 is an average of the Ktrans time courses from all the subjects with adjacent curves indicating the plus and minus one standard error of the mean curves. This figure demonstrates the peak Ktrans value immediately after the LITT procedure with persistent elevation out to about 4 weeks. Radiographically, persistent contrast enhancement and FLAIR hyperintensity were observed well past 6 weeks and in many cases more than 10 weeks later. Several patients had recurrent tumor by radiographic criteria (increasing size of the edema and enhancing area around the tumor site) and these patients also demonstrated a corresponding increase in the Ktrans value. These recurrences occurred after the 10-week mark and thus were not included in Fig 3. Importantly no difference in the pattern of Ktrans tracing was consistently observed between the 10 patients receiving late doxorubicin treatment and the 4 patients receiving early doxorubicin treatment. In summary, these results indicate that the peritumoral BBB disruption as measured by Ktrans peaked immediately after LITT and persisted above baseline for an additional 4 weeks.

……

To optimize the ELISA assay for BSE, we collected sera from 3 patients with a newly diagnosed low-grade (WHO grade 2) glioma before and after their planned craniotomy and surgical resection, and determined serum concentrations of BSE. WHO grade 2 gliomas were chosen for the optimization because as they are generally non-contrast enhanced tumors on brain MRI, tumor-associated BBB is relatively intact and consequently, serum concentrations of brain-specific factors are predicted to be low pre-operatively and to then rise post-operatively due to the BBB compromise from the surgery. Serum BSE concentrations were low prior to surgery and then, as predicted, consistently increased after open craniotomy and tumor resection, thus indicating that this method had adequate sensitivity in detecting changes in serum levels of BSE due to disruption of the BBB (Fig 4).

Fig 4. Optimization of the BSE ELISA assay for measuring BBB disruption.

Serum concentrations of BSE before and after open craniotomy for surgical debulking in 3 subjects (A, B, and C) with a low-grade glioma, WHO grade II. *p<0.05.  http://dx.doi.org:/10.1371/journal.pone.0148613.g004

……

Fig 5. BBB disruption induced by LITT as measured by serum biomarkers
Serum concentrations of BSE for each of the 14 evaluable subjects in the study (A-N) and as the mean + SEM (O) as a function of time in days from the LITT procedure. In 7/14 subjects, serum BSE levels slightly decreased immediately after LITT, then in 13/14 subjects, serum BSE levels rose shortly after LITT, peaked between 1–3 weeks after LITT, and then decreased by the 6-week time point. In Patient #12, serum BSE concentration increased at week 10 coincident with an increased Ktrans at the same time point, consistent with a recurrent tumor as demonstrated on diagnostic MR imaging. Patient #15’s serum BSE concentration began to rise by week 4, consistent with early multifocal recurrent disease as demonstrated on diagnostic MR imaging.  http://dx.doi.org:/10.1371/journal.pone.0148613.g005
…….

LITT is a minimally invasive neurosurgical technique that achieves effective tumor cytoreduction of brain tumors using a laser to deliver hyperthermic ablation. Here we have demonstrated that an unexpected, potentially useful effect of LITT is its ability to also disrupt the BBB in the peritumoral region that extends outwards 1–2 cm from the viable tumor rim. Importantly, the disruption persists in all 14 evaluable, treated patients for up to 4 weeks after LITT as measured quantitatively by DCE-MRI and up to 6 weeks as measured by serum levels of the brain-specific factor BSE. These observations indicate that after LITT there is a window during which enhanced local delivery of therapeutic agents into the desired location (i.e. peritumoral region) can potentially be achieved.

In all of the patients in this series, the peaks of serum concentrations of BSE showed wider variations and were delayed from several days to 1–2 weeks following the peak of BBB disruption as measured by Ktrans. The wider variations and delay of BSE concentrations lead to relatively low correlation coefficients between the two parameters and could be explained by: 1) the higher data point resolution for the serum values versus DCE-MRI values (weekly versus biweekly, respectively); 2) interval physiologic breakdown of thermally ablated tissue coupled with subsequent diffusion and equilibration between the intracranial and peripheral compartments; and 3) high inter-tumor heterogeneity among patients resulting in a wide variation in the rates at which ablated tissues of different compositions are broken down and released into the circulation. Whether these differences may be in part due to tumor-related factors such as IDH1/2 mutations and MGMT promoter methylation is unclear due to the small number of subjects. More importantly, both methods showed that the peritumoral BBB disruption induced by LITT was temporary, decreasing soon after peaking and being resolved by 4–6 weeks in most patients. In addition, although no significant difference in all the BBB measurement parameters was observed between the early and late doxorubicin treatment arms, the number of evaluable subjects was too small to allow generalization at this time.

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Brain Cancer Vaccine in Development and other considerations

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

GEN News Highlights   Mar 3, 2016

Advanced Immunotherapeutic Method Shows Promise against Brain Cancer

http://www.genengnews.com/gen-news-highlights/advanced-immunotherapeutic-method-shows-promise-against-brain-cancer/81252433/

 

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The researchers induced a specific type of cell death in brain cancer cells from mice. The dying cancer cells were then incubated together with dendritic cells, which play a vital role in the immune system. The researchers discovered that this type of cancer cell killing releases “danger signals” that fully activate the dendritic cells. “We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” Professor Patrizia Agostinis explains. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.” [©KU Leuven Laboratory of Cell Death Research & Therapy, Dr. Abhishek D. Garg]

 

Scientists from KU Leuven in Belgium say they have shown that next-generation cell-based immunotherapy may offer new hope in the fight against brain cancer.

Cell-based immunotherapy involves the injection of a therapeutic anticancer vaccine that stimulates the patient’s immune system to attack the tumor. Thus far, the results of this type of immunotherapy have been mildly promising. However, Abhishek D. Garg and Professor Patrizia Agostinis from the KU Leuven department of cellular and molecular medicine believe they have found a novel way to produce more effective cell-based anticancer vaccines.

The researchers induced a specific type of cell death in brain cancer cells from mice. The dying cancer cells were then incubated together with dendritic cells, which play a vital role in the immune system. The investigators discovered that this type of cancer cell killing releases “danger signals” that fully activate the dendritic cells.

“We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” explains Prof. Agostinis. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.”

Combined with chemotherapy, this novel cell-based immunotherapy drastically increased the survival rates of mice afflicted with brain tumors. Almost 50% of the mice were completely cured. None of the mice treated with chemotherapy alone became long-term survivors.

“The major goal of any anticancer treatment is to kill all cancer cells and prevent any remaining malignant cells from growing or spreading again,” says Professor Agostinis. “This goal, however, is rarely achieved with current chemotherapies, and many patients relapse. That’s why the co-stimulation of the immune system is so important for cancer treatments. Scientists have to look for ways to kill cancer cells in a manner that stimulates the immune system. With an eye on clinical studies, our findings offer a feasible way to improve the production of vaccines against brain tumors.”

The team published its study (“Dendritic Cell Vaccines Based on Immunogenic Cell Death Elicit Danger Signals and T Cell–Driven Rejection of High-Grade Glioma”) in Science Translational Medicine.

 

Dendritic cell vaccines based on immunogenic cell death elicit danger signals and T cell–driven rejection of high-grade glioma

 

SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma

 

Cortical GABAergic excitation contributes to epileptic activities around human glioma

 

Spherical Nucleic Acid Nanoparticle Conjugates as an RNAi-Based Therapy for Glioblastoma

Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many “undruggable” oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)–based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)—an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas—were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.

 

Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy

Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

 

Neural Stem Cell–Mediated Enzyme/Prodrug Therapy for Glioma: Preclinical Studies

 

Magnetic Resonance Metabolic Imaging of Glioma

 

Exploiting the Immunogenic Potential of Cancer Cells for Improved Dendritic Cell Vaccines

Cancer immunotherapy is currently the hottest topic in the oncology field, owing predominantly to the discovery of immune checkpoint blockers. These promising antibodies and their attractive combinatorial features have initiated the revival of other effective immunotherapies, such as dendritic cell (DC) vaccinations. Although DC-based immunotherapy can induce objective clinical and immunological responses in several tumor types, the immunogenic potential of this monotherapy is still considered suboptimal. Hence, focus should be directed on potentiating its immunogenicity by making step-by-step protocol innovations to obtain next-generation Th1-driving DC vaccines. We review some of the latest developments in the DC vaccination field, with a special emphasis on strategies that are applied to obtain a highly immunogenic tumor cell cargo to load and to activate the DCs. To this end, we discuss the effects of three immunogenic treatment modalities (ultraviolet light, oxidizing treatments, and heat shock) and five potent inducers of immunogenic cell death [radiotherapy, shikonin, high-hydrostatic pressure, oncolytic viruses, and (hypericin-based) photodynamic therapy] on DC biology and their application in DC-based immunotherapy in preclinical as well as clinical settings.

Cancer immunotherapy has gained considerable momentum over the past 5 years, owing predominantly to the discovery of immune checkpoint inhibitors. These inhibitors are designed to release the brakes of the immune system that under physiological conditions prevent auto-immunity by negatively regulating cytotoxic T lymphocyte (CTL) function. Following the FDA approval of the anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) monoclonal antibody (mAb) ipilimumab (Yervoy) in 2011 for the treatment of metastatic melanoma patients (1), two mAbs targeting programed death (PD)-1 receptor signaling (nivolumab and pembrolizumab) have very recently joined the list of FDA-approved checkpoint blockers (respectively, for the treatment of metastatic squamous non-small cell lung cancer and relapsed/refractory melanoma patients) (2, 3).

However, the primary goal of cancer immunotherapy is to activate the immune system in cancer patients. This requires the induction of tumor-specific T-cell-mediated antitumor immunity. Checkpoint blockers are only able to abrogate the brakes of a functioning antitumoral immune response, implying that only patients who have pre-existing tumor-specific T cells will benefit most from checkpoint blockade. This is evidenced by the observation that ipilimumab may be more effective in patients who have pre-existing, albeit ineffective, antitumor immune responses (4). Hence, combining immune checkpoint blockade with immunotherapeutic strategies that prime tumor-specific T cell responses might be an attractive and even synergistic approach. This relatively new paradigm has lead to the revival of existing, and to date disappointing (as monotherapies), active immunotherapeutic treatment modalities. One promising strategy to induce priming of tumor-specific T cells is dendritic cell (DC)-based immunotherapy.

Dendritic cells are positioned at the crucial interface between the innate and adaptive immune system as powerful antigen-presenting cells capable of inducing antigen-specific T cell responses (5). Therefore, they are the most frequently used cellular adjuvant in clinical trials. Since the publication of the first DC vaccination trial in melanoma patients in 1995, the promise of DC immunotherapy is underlined by numerous clinical trials, frequently showing survival benefit in comparison to non-DC control groups (68). Despite the fact that most DC vaccination trials differ in several vaccine parameters (i.e., site and frequency of injection, nature of the DCs, choice of antigen), DC vaccination as a monotherapy is considered safe and rarely associates with immune-related toxicity. This is in sharp contrast with the use of mAbs or cytokine therapies. Ipilumumab has, for instance, been shown to induce immune-related serious adverse events in up to one-third of treated melanoma patients (1). The FDA approval of Sipuleucel-T (Provenge), an autologous DC-enriched vaccine for hormone-resistant metastatic prostate cancer, in 2010 is really considered as a milestone in the vaccination community (9). After 15 years of extensive clinical research, Sipileucel-T became the first cellular immunotherapy ever that received FDA approval, providing compelling evidence for the substantial socio-economic impact of DC-based immunotherapy. DC vaccinations have most often been applied in patients with melanoma, prostate cancer, high-grade glioma, and renal cell cancer. Although promising objective responses and tumor-specific T cell responses have been observed in all these cancer-types (providing proof-of-principle for DC-based immunotherapy), the clinical success of this treatment is still considered suboptimal (6). This poor clinical efficacy can in part be attributed to the severe tumor-induced immune suppression and the selection of patients with advanced disease status and poor survival prognostics (6, 1012).

There is a consensus in the field that step-by-step optimization and standardization of the production process of DC vaccines, to obtain a Th1-driven immune response, might enhance their clinical efficacy (13). In this review, we address some recent DC vaccine adaptations that impact DC biology. Combining these novel insights might bring us closer to an ideal DC vaccine product that can trigger potent CTL- and Th1-driven antitumor immunity.

One factor requiring more attention in this production process is the immunogenicity of the dying or dead cancer cells used to load the DCs. It has been shown in multiple preclinical cancer models that the methodology used to prepare the tumor cell cargo can influence the in vivo immunogenic potential of loaded DC vaccines (1419). Different treatment modalities have been described to enhance the immunogenicity of cancer cells in the context of DC vaccines. These treatments can potentiate antitumor immunity by inducing immune responses against tumor neo-antigens and/or by selectively increasing the exposure/release of particular damage-associated molecular patterns (DAMPs) that can trigger the innate immune system (14, 1719). The emergence of the concept of immunogenic cell death (ICD) might even further improve the immunogenic potential of DC vaccines. Cancer cells undergoing ICD have been shown to exhibit excellent immunostimulatory capacity owing to the spatiotemporally defined emission of a series of critical DAMPs acting as potent danger signals (20, 21). Thus far, three DAMPs have been attributed a crucial role in the immunogenic potential of nearly all ICD inducers: the surface-exposed “eat me” signal calreticulin (ecto-CRT), the “find me” signal ATP and passively released high-mobility group box 1 (HMGB1) (21). Moreover, ICD-experiencing cancer cells have been shown in various mouse models to act as very potent Th1-driving anticancer vaccines, already in the absence of any adjuvants (21, 22). The ability to reject tumors in syngeneic mice after vaccination with cancer cells (of the same type) undergoing ICD is a crucial hallmark of ICD, in addition to the molecular DAMP signature (21).

Here, we review the effects of three frequently used immunogenic modalities and four potent ICD inducers on DC biology and their application in DC vaccines in preclinical as well as clinical settings (Tables (Tables11 and and2).2). Moreover, we discuss the rationale for combining different cell death-inducing regimens to enhance the immunogenic potential of DC vaccines and to ensure the clinical relevance of the vaccine product.

A list of prominent enhancers of immunogenicity and ICD inducers applied in DC vaccine setups and their associations with DAMPs and DC biology.
A list of preclinical tumor models and clinical studies for evaluation of the in vivo potency of DC vaccines loaded with immunogenically killed tumor cells.
The Impact of DC Biology on the Efficacy of DC Vaccines

Over the past years, different DC vaccine parameters have been shown to impact the clinical effectiveness of DC vaccinations. In the next section, we will elaborate on some promising adaptations of the DC preparation protocol.

Given the labor-intensive ex vivo culturing protocol of monocyte-derived DCs and inspired by the results of the Provenge study, several groups are currently exploiting the use of blood-isolated naturally circulating DCs (7678). In this context, De Vries et al. evaluated the use of antigen-loaded purified plasmacytoid DCs for intranodal injection in melanoma patients (79). This strategy was feasible and induced only very mild side effects. In addition, the overall survival of vaccinated patients was greatly enhanced as compared to historical control patients. However, it still remains to be determined whether this strategy is more efficacious than monocyte-derived DC vaccine approaches (78). By contrast, experiments in the preclinical GL261 high-grade glioma model recently showed that vaccination with tumor antigen-loaded myeloid DCs resulted in more robust Th1 responses and a stronger survival benefit as compared to mice vaccinated with their plasmacytoid counterparts (80).

In view of their strong potential to stimulate cytotoxic T cell responses, several groups are currently exploring the use of Langerhans cell-like DCs as sources for DC vaccines (8183). These so-called IL-15 DCs can be derived from CD14+monocytes by culturing them with IL-15 (instead of the standard IL-4). Recently, it has been shown that in comparison to IL-4 DCs, these cells have an increased capacity to stimulate antitumor natural killer (NK) cell cytotoxicity in a contact- and IL-15-dependent manner (84). NK cells are increasingly being recognized as crucial contributors to antitumor immunity, especially in DC vaccination setups (85, 86). Three clinical trials are currently evaluating these Langerhans cell-type DCs in melanoma patients (NCT00700167, NCT 01456104, and NCT01189383).

Targeting cancer stem cells is another promising development, particularly in the setting of glioma (87). Glioma stem cells can foster tumor growth, radio- and chemotherapy-resistance, and local immunosuppression in the tumor microenvironment (87, 88). Furthermore, glioma stem cells may express higher levels of tumor-associated antigens and MHC complex molecules as compared to non-stem cells (89, 90). A preclinical study in a rodent orthotopic glioblastoma model has shown that DC vaccines loaded with neuropsheres enriched in cancer stem cells could induce more immunoreactivity and survival benefit as compared to DCs loaded with GL261 cells grown under standard conditions (91). Currently there are four clinical trials ongoing in high-grade glioma patients evaluating this approach (NCT00890032, NCT00846456, NCT01171469, and NCT01567202).

With regard to the DC maturation status of the vaccine product, a phase I/II clinical trial in metastatic melanoma patients has confirmed the superiority of mature antigen-loaded DCs to elicit immunological responses as compared to their immature counterparts (92). This finding was further substantiated in patients diagnosed with prostate cancer and recurrent high-grade glioma (93, 94). Hence, DCs need to express potent costimulatory molecules and lymph node homing receptors in order to generate a strong T cell response. In view of this finding, the route of administration is another vaccine parameter that can influence the homing of the injected DCs to the lymph nodes. In the context of prostate cancer and renal cell carcinoma it has been shown that vaccination routes with access to the draining lymph nodes (intradermal/intranodal/intralymphatic/subcutaneous) resulted in better clinical response rates as compared to intravenous injection (93). In melanoma patients, a direct comparison between intradermal vaccination and intranodal vaccination concluded that, although more DCs reached the lymph nodes after intranodal vaccination, the melanoma-specific T cells induced by intradermal vaccination were more functional (95). Furthermore, the frequency of vaccination can also influence the vaccine’s immunogenicity. Our group has shown in a cohort-comparison trial involving relapsed high-grade glioma patients that shortening the interval between the four inducer DC vaccines improved the progression-free survival curves (58, 96).

Another variable that has been systematically studied is the cytokine cocktail that is applied to mature the DCs. The current gold standard cocktail for DC maturation contains TNF-α, IL-1β, IL-6, and PGE2 (97, 98). Although this cocktail upregulates DC maturation markers and the lymph node homing receptor CCR7, IL-12 production by DCs could not be evoked (97, 98). Nevertheless, IL-12 is a critical Th1-driving cytokine and DC-derived IL-12 has been shown to associate with improved survival in DC vaccinated high-grade glioma and melanoma patients (99, 100). Recently, a novel cytokine cocktail, including TNF-α, IL-1β, poly-I:C, IFN-α, and IFN-γ, was introduced (101, 102). The type 1-polarized DCs obtained with this cocktail produced high levels of IL-12 and could induce strong tumor-antigen-specific CTL responses through enhanced induction of CXCL10 (99). In addition, CD40-ligand (CD40L) stimulation of DCs has been used to mature DCs in clinical trials (100, 103). Binding of CD40 on DCs to CD40L on CD4+ helper T cells licenses DCs and enables them to prime CD8+ effector T cells.

A final major determinant of the vaccine immunogenicity is the choice of antigen to load the DCs. Two main approaches can be applied: loading with selected tumor antigens (tumor-associated antigens or tumor-specific antigens) and loading with whole tumor cell preparations (13). The former strategy enables easier immune monitoring, has a lower risk of inducing auto-immunity, and can provide “off-the-shelf” availability of the antigenic cargo. Whole tumor cell-based DC vaccines, on the other hand, are not HLA-type dependent, have a reduced risk of inducing immune-escape variants, and can elicit immunity against multiple tumor antigens. Meta-analytical data provided by Neller et al. have demonstrated enhanced clinical efficacy in several tumor types of DCs loaded with whole tumor lysate as compared to DCs pulsed with defined tumor antigens (104). This finding was recently also substantiated in high-grade glioma patients, although this study was not set-up to compare survival parameters (105).

Toward a More Immunogenic Tumor Cell Cargo

The majority of clinical trials that apply autologous whole tumor lysate to load DC vaccines report the straightforward use of multiple freeze–thaw cycles to induce primary necrosis of cancer cells (8, 93). Freeze–thaw induced necrosis is, however, considered non-immunogenic and has even been shown to inhibit toll-like receptor (TLR)-induced maturation and function of DCs (16). To this end, many research groups have focused on tackling this roadblock by applying immunogenic modalities to induce cell death.

Immunogenic Treatment Modalities

Tables Tables11 and and22 list some frequently applied treatment methods to enhance the immunogenic potential of the tumor cell cargo that is used to load DC vaccines in an ICD-independent manner (i.e., these treatments do not meet the molecular and/or cellular determinants of ICD). Immunogenic treatment modalities can positively impact DC biology by inducing particular DAMPs in the dying cancer cells (Table (Table1).1). Table Table22 lists the preclinical and clinical studies that investigated their in vivo potential. Figure Figure11 schematically represents the application and the putative modes of action of these immunogenic enhancers in the setting of DC vaccines.

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A schematic representation of immunogenic DC vaccines. Cancer cells show enhanced immunogenicity upon treatment with UV irradiation, oxidizing treaments, and heat shock, characterized by the release of particular danger signals and the (increased) production of tumor (neo-)antigens. Upon loading onto DCs, DCs undergo enhanced phagocytosis and antigen uptake and show phenotypic and partial functional maturation. Upon in vivo immunization, these DC vaccines elicit Th1- and cytotoxic T lymphocyte (CTL)-driven tumor rejection.

Ultraviolet Irradiation ….

Oxidation-Inducing Modalities

In recent years, an increasing number of data were published concerning the ability of oxidative stress to induce oxidation-associate molecular patterns (OAMPs), such as reactive protein carbonyls and peroxidized phospholipids, which can act as DAMPs (28, 29) (Table (Table1).1). Protein carbonylation, a surrogate indicator of irreversible protein oxidation, has for instance been shown to improve cancer cell immunogenicity and to facilitate the formation of immunogenic neo-antigens (30, 31).

One prototypical enhancer of oxidation-based immunogenicity is radiotherapy (21,23). In certain tumor types, such as high-grade glioma and melanoma, clinical trials that apply autologous whole tumor lysate to load DC vaccines report the random use of freeze–thaw cycles (to induce necrosis of cancer cells) or a combination of freeze–thaw cycles and subsequent high-dose γ-irradiation (8, 18) (Table (Table2).2). However, from the available clinical evidence, it is unclear which of both methodologies has superior immunogenic potential. In light of the oxidation-based immunogenicity that is associated with radiotherapy, we recently demonstrated the superiority of DC vaccines loaded with irradiated freeze–thaw lysate (in comparison to freeze–thaw lysate) in terms of survival advantage in a preclinical high-grade glioma model (18) (Table (Table2).2). ….

Heat Shock Treatment

Heat shock is a term that is applied when a cell is subjected to a temperature that is higher than that of the ideal body temperature of the organisms of which the cell is derived. Heat shock can induce apoptosis (41–43°C) or necrosis (>43°C) depending on the temperature that is applied (110). The immunogenicity of heat shock treated cancer cells largely resides within their ability to produce HSPs, such as HSP60, HSP70, and HSP90 (17, 32) (Table (Table1).1). …

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http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=4712296_fimmu-06-00663-g002.jpg

Figure 2

A schematic representation of immunogenic cell death (ICD)-based DC vaccines. ICD causes cancer cells to emit a spatiotemporally defined pattern of danger signals. Upon loading of these ICD-undergoing cancer cells onto DCs, they induce extensive phagocytosis and antigen uptake. Loaded DCs show enhanced phenotypic and functional maturation and immunization with these ICD-based DC vaccines instigates Th1-, Th17-, and cytotoxic T lymphocyte (CTL)-driven antitumor immunity in vivo.
Inducers of Immunogenic Cell Death

Immunogenic cell death is a cell death regimen that is associated with the spatiotemporally defined emission of immunogenic DAMPs that can trigger the immune system (20, 21, 113). ICD has been found to depend on the concomitant induction of reactive oxygen species (ROS) and activation of endoplasmatic reticulum (ER) stress (111). Besides the three DAMPs that are most crucial for ICD (ecto-CRT, ATP, and HMGB1), other DAMPs such as surface-exposed or released HSPs (notably HSP70 and HSP90) have also been shown to contribute to the immunogenic capacity of ICD inducers (20, 21). The binding of these DAMPs to their respective immune receptors (CD91 for HSPs/CRT, P2RX7/P2RY2 for ATP, and TLR2/4 for HMGB1/HSP70) leads to the recruitment and/or activation of innate immune cells and facilitates the uptake of tumor antigens by antigen-presenting cells and their cross-presentation to T cells eventually leading to IL-1β-, IL-17-, and IFN-γ-dependent tumor eradiation (22). This in vivo tumor rejecting capacity induced by dying cancer cells in the absence of any adjuvant, is considered as a prerequisite for an agent to be termed an ICD inducer. …

Although the list of ICD inducers is constantly growing (113), only few of these immunogenic modalities have been tested in order to generate an immunogenic tumor cell cargo to load DC vaccines (Tables (Tables11 and and2).2). Figure Figure22 schematically represents the preparation of ICD-based DC vaccines and their putative modes of action.

Radiotherapy

Ionizing X-ray or γ-ray irradiation exerts its anticancer effect predominantly via its capacity to induce DNA double-strand breaks leading to intrinsic cancer cell apoptosis (114). The idea that radiotherapy could also impact the immune system was derived from the observation that radiotherapy could induce T-cell-mediated delay of tumor growth in a non-irradiated lesion (115). This abscopal (ab-scopus, away from the target) effect of radiotherapy was later explained by the ICD-inducing capacity (116). Together with anthracyclines, γ-irradiation was one of the first treatment modalities identified to induce ICD. …

Shikonin

The phytochemical shikonin, a major component of Chinese herbal medicine, is known to inhibit proteasome activity. It serves multiple biological roles and can be applied as an antibacterial, antiviral, anti-inflammatory, and anticancer treatment. …

High-hydrostatic pressure

High-hydrostatic pressure (HHP) is an established method to sterilize pharmaceuticals, human transplants, and food. HHP between 100 and 250 megapascal (MPa) has been shown to induce apoptosis of murine and human (cancer) cells (121123). While DNA damage does not seem to be induced by HHP <1000 MPa, HHP can inhibit enzymatic functions and the synthesis of cellular proteins (122). Increased ROS production was detected in HHP-treated cancer cell lines and ER stress was evidenced by the rapid phosphorylation of eIF2α (42).  …

Oncolytic Viruses

Oncolytic viruses are self-replicating, tumor selective virus strains that can directly lyse tumor cells. Over the past few years, a new oncolytic paradigm has risen; entailing that, rather than utilizing oncolytic viruses solely for direct tumor eradication, the cell death they induce should be accompanied by the elicitation of antitumor immune responses to maximize their therapeutic efficacy (128). One way in which these oncolytic viruses can fulfill this oncolytic paradigm is by inducing ICD (128).

Thus far, three oncolytic virus strains can meet the molecular requirements of ICD; coxsackievirus B3 (CVB3), oncolytic adenovirus and Newcastle disease virus (NDV) (Table (Table1)1) (113). Infection of tumor cells with these viruses causes the production of viral envelop proteins that induce ER stress by overloading the ER. Hence, all three virus strains can be considered type II ICD inducers (113). …

Photodynamic therapy

Photodynamic therapy (PDT) is an established, minimally invasive anticancer treatment modality. It has a two-step mode of action involving the selective uptake of a photosensitizer by the tumor tissue, followed by its activation by light of a specific wavelength. This activation results in the photochemical production of ROS in the presence of oxygen (129131). One attractive feature of PDT is that the ROS-based oxidative stress originates in the particular subcellular location where the photosensitizer tends to accumulate, ultimately leading to the destruction of the tumor cell (132). …

Combinatorial Regimens

In DC vaccine settings, cancer cells are often not killed by a single treatment strategy but rather by a combination of treatments. In some cases, the underlying rationale lies within the additive or even synergistic value of combining several moderately immunogenic modalities. The combination of radiotherapy and heat shock has, for instance, been shown to induce higher levels of HSP70 in B16 melanoma cells than either therapy alone (16). In addition, a combination therapy consisting of heat shock, γ-irradiation, and UV irradiation has been shown to induce higher levels of ecto-CRT, ecto-HSP90, HMGB1, and ATP in comparison to either therapy alone or doxorubicin, a well-recognized inducer of ICD (57). ….

Triggering antitumor immune responses is an absolute requirement to tackle metastatic and diffusely infiltrating cancer cells that are resistant to standard-of-care therapeutic regimens. ICD-inducing modalities, such as PDT and radiotherapy, have been shown to be able to act as in situ vaccines capable of inducing immune responses that caused regression of distal untreated tumors. Exploiting these ICD inducers and other immunogenic modalities to obtain a highly immunogenic antigenic tumor cell cargo for loading DC vaccines is a highly promising application. In case of the two prominent ICD inducers, Hyp-PDT and HHP, preclinical studies evaluating this relatively new approach are underway and HHP-based DC vaccines are already undergoing clinical testing. In the preclinical testing phase, more attention should be paid to some clinically driven considerations. First, one should consider the requirement of 100% mortality of the tumor cells before in vivo application. A second consideration from clinical practice (especially in multi-center clinical trials) is the fact that most tumor specimens arrive in the lab in a frozen state. This implies that a significant number of cells have already undergone non-immunogenic necrosis before the experimental cell killing strategies are applied. ….

 

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3-D molecular structures

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New method enables discovery of 3D structures for molecules important to medicine

February 19, 2016  http://www.kurzweilai.net/new-method-enables-discovery-of-3d-structures-for-molecules-important-to-medicine

 

If you zap a crystal (green, left) containing highly ordered protein molecules with X-rays, the X-rays scatter and produce useful regular patterns of spots known as Bragg peaks (red dots). But if the protein in the crystal is less ordered or disordered (right), the X-rays produce some spots along with patterns of light and shade known as a continuous diffraction pattern that’s not useful. (credit: Eberhard Reimann/DESY)

Researchers have overcome a long-standing technical barrier to imaging 3D structures of thousands of molecules important to medicine and biology.

The 3D structures of many protein molecules have been discovered using a technique called X-ray crystallography, but the method relies on scientists being able to produce highly ordered crystals containing the protein molecules in a regular arrangement. When X-rays are shone on highly ordered crystals, the X-rays scatter and produce regular patterns of spots called Bragg peaks (see figure above, left). High-quality Bragg peaks contain the information to produce high-resolution 3D structures of proteins.

Unfortunately, many important and complex biomolecules do not form highly ordered crystals; instead, the protein arrangements are slightly disordered. When X-rays are shone on these more disordered crystals, a smaller number of Bragg peaks are produced, along with a vague pattern of light and shadow known as a continuous diffraction pattern (right).

In the past, scientists discarded these less-than-perfect crystals. Unfortunately, many of the molecules forming disordered crystals are important molecular complexes such as those that span cell membranes.

X-raying crystal patterns to detect hidden protein structures

Analysis of Bragg peaks alone (top) reveals far less details than analysis of the high-res continuous diffraction pattern (bottom). Magnifying glasses show actual data. (credit: DESY, Eberhard Reimann)

So a team led by Professor Henry Chapman from the Center for Free-Electron Laser Science at DESY in Hamburg, Germany turned to the world’s most powerful X-ray laser: the SLAC LCLS at Stanford University.

Kartik Ayyer, PhD., lead author of the article in Nature, explains that the method uses an approach similar to that used to image a single molecule.

“If you would shoot X-rays on a single molecule, it would produce a continuous diffraction pattern free of any Bragg spots,” he says. “The pattern would be extremely weak, however, and very difficult to measure. But the ‘background’ in our crystal analysis is like accumulating many shots from individually aligned single molecules. We essentially just use the crystal as a way to get a lot of single molecules, aligned in common orientations, into the beam.”

As the model protein, the researchers crystallized photosystem II (PSII), a large membrane–protein complex of photosynthesis that plants use to produce oxygen for life on Earth.

After exposing the crystal to X-rays, the researchers first analyzed the Bragg peaks of PSII to produce a low-resolution outline of the 3D structure (figure above, top). They then improved this data, using an algorithm, to analyze the continuous diffraction pattern and produced a higher-resolution 3D structure (figure, bottom).

This novel method means that imperfect crystals containing a slightly disordered protein arrangement can now be used to “directly view large protein complexes in atomic detail,” says Chapman. “This kind of continuous diffraction has actually been seen for a long time from many different poorly diffracting crystals,” says Chapman. “It wasn’t understood that you can get structural information from it and so analysis techniques suppressed it.

“We’re going to be busy to see if we can solve [additional] structures of molecules from old discarded data.”


Abstract of Macromolecular diffractive imaging using imperfect crystals

The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins—they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5-ångström limit of measurable Bragg peaks, which allows us to phase the pattern directly. Using the molecular envelope conventionally determined at 4.5 ångströms as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 ångströms. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing.

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Tunable light sources

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Putting Tunable Light Sources to the Test

Common goals of spectroscopy applications such as studying the chemical or biological properties of a material often dictate the requirements of the measurement system’s lamp, power supply and monochromator.

JEFF ENG AND JOHN PARK, PH.D., NEWPORT CORP.   http://www.photonics.com/Article.aspx?AID=58302

Many common spectroscopic measurements require the coordinated operation of a detection instrument and light source, as well as data acquisition and processing. Integration of individual components can be challenging and various applications may have different requirements. Conventional lamp-based tunable light sources are a popular choice for applications requiring a measurement system with this degree of capability.

Many types of tunable light sources are available, with differences in individual component performance translating to the performance of the system as a whole. Tunable light sources are finding themselves to be an especially ideal system for one application in particular: quantum efficiency and spectral responsivity characterization of photonic sensors, such as solar cells.

Xenon and mercury xenon lamps, two examples of DC arc lamps.

http://www.photonics.com/images/Web/Articles/2016/2/10/Light_Lamps.jpg

Xenon and mercury xenon lamps, two examples of DC arc lamps.

The tunable light source’s (TLS) versatility as both a broadband and high-resolution monochromatic light source makes the unit suitable for a variety of applications, such as the study of wavelength-dependent chemical or biological properties or wavelength-induced physical changes of materials. These light sources can also be used in color analysis and reflectivity measurements of materials for quality purposes.

Among their unique attributes, the TLS can produce monochromatic light from the UV to near-infrared (NIR). Lamp-based TLSs feature two major components: a light source and a monochromator. Common lamps used in TLSs are the DC arc lamp and quartz tungsten halogen (QTH) lamp. While both of these lamps have a broad emission spectrum, arc and QTH lamps differ in the characteristic wavelength emissions or relatively smooth shape of their spectral output curves, respectively. A stable power supply for the lamp is a critical component since most applications require high light output power stability1.

Smooth spectral output vs. monochromator throughput

DC arc lamps are excellent sources of continuous wave, broadband light. They consist of two electrodes (an anode and a cathode) separated by a gas such as neon, argon, mercury or xenon. Light is generated by ionizing the gas between the electrodes. The bright broadband emission from this short arc between the anode and cathode makes these lamps high-intensity point sources, capable of being collimated with the proper lens configuration.

DC arc lamps also offer the advantages of long lifetime, superior monochromator throughput (particularly in the UV range) and a smaller divergence angle. They are particularly well-suited for fiber coupling applications2. (See Figure 1.)

A xenon arc lamp housed in an Oriel Research lamp housing.

Figure 1. A xenon arc lamp housed in an Oriel Research lamp housing. Photo courtesy of Newport Corp.

Xenon (Xe) arc lamps, in particular, have a relatively smooth emission curve in the UV to visible spectrums, with characteristic wavelengths emitted from 380 to 750 nm. However, strong xenon peaks are emitted between 750 to 1000 nm.

Their sunlike emission spectrum and about 5800 K color temperature make them a popular choice for solar simulation applications. (See Figure 2.)

Arc lamps can have the following specialty characteristics:

Ozone-free: Wavelength emissions below about 260 nm create toxic ozone. Ideally, an arc lamp is operated outdoors or in a room with adequate ventilation to protect the user from the ozone created.

UV-enhanced: For applications requiring additional UV light intensity, UV-enhanced lamps should be used. These lamps provide the same visible to NIR performance of an arc lamp while providing high-intensity UV output due to changes in the material of the lamp’s glass envelope.

High-stability: High-stability arc lamps are made of a higher quality cathode than that typically used for arc lamp construction. As a result, no arc wander occurs, allowing the lamp to maintain consistent output intensity throughout its lifetime.

The spectral output of 3000-W Xe and 250-W QTH lamps used in Oriel’s Tunable Light Sources.

Figure 2. The spectral output of 3000-W Xe and 250-W QTH lamps used in Oriel’s Tunable Light Sources.Photo courtesy of Newport Corp.

QTH lamps produce light by heating a filament wire with an electric current. The hot filament wire is surrounded by a vacuum or inert gas to prevent oxidation. QTH lamps are not very efficient at converting electricity to light, but they offer very accurate color reproduction due to their continuous blackbody spectrum. These lamps are a popular alternative to arc lamps due to their higher output intensity stability and lack of intense UV light emission, spectral emission lines in their output curve and toxic ozone production. These advantages over traditional DC arc lamps make QTH lamps preferable for radiometric and photometric applications as well as excitation sources of visible to NIR light. QTH lamps are also easier to handle and install, and produce a smooth output spectrum. Selecting the most appropriate lamp type is a matter of deciding which performance criteria are most important.

Constant current vs. constant power

The power supply is a vital component for operating a DC arc or QTH lamp with minimum light ripple. The lamps are operated in either constant current or constant power mode and are used in applications such as radiometric measurements, where a stable light output is required for accurate measurement. Providing stable electrical power to the lamp is important since fluctuations in the wavelength and output intensity of the light source impact the accuracy of measurement.

There is very little difference in the short-term output stability when operating an arc lamp or QTH lamp in constant current or constant power mode. However, the differences appear as the lamp ages. For arc lamps, even with a stable power supply, deposits on the inside of the lamp envelope are visible as the electrodes degrade, which causes an unstable arc position, changing the electrical characteristics of the arc lamp. The distance between the cathode and anode of the arc lamp increases, raising the lamp’s operating voltage. For QTH lamps, deposits on the inside of the lamp envelope are visible as the lamp filament degrades, changing the electrical and spectral characteristics of the lamp.

In power mode, the lamp is operated at a constant power setting. As the voltage cannot be changed, the current is raised or lowered to maintain the power at the same level. As the lamp ages, the radiant output decreases. However, lamp lifetime is prolonged.

In current mode, the lamp is operated at a constant current setting. As the voltage cannot be changed, the power is raised or lowered to maintain the current at the same level. As the lamp ages, the input power required for operation is increased. This results in greater output power, which, to some extent, may help compensate for a darkening lamp envelope. However, the lamp’s lifetime is greatly reduced due to the increase in power.

Although power supplies are highly regulated, there are factors beyond the control of the power supply that may affect light output. Some of these factors include lamp aging, ambient temperature fluctuations and filament erosion. For applications in which high stability light output intensity is especially critical, optical feedback control of power supply is suggested in order to compensate for such factors3. (See Figure 3.)

Oriel’s OPS Series Power Supplies offer the option of operating a lamp in constant power, constant current or intensity operation modes.

Figure 3. Oriel’s OPS Series Power Supplies offer the option of operating a lamp in constant power, constant current or intensity operation modes. Photo courtesy of Newport Corp.

Diffraction gratings narrow the wavelength band

Monochromators use diffraction gratings to spatially isolate and select a narrow band of wavelengths from a wider wavelength emitting light source. They are a valuable piece of equipment because they can be used to create quasi-monochromatic light and also take high precision spectral measurements. A high precision stepper motor is typically used to select the desired wavelength and switch between diffraction gratings quickly, without sacrificing instrument performance.

Determining which slit width to use is based on the trade-off between light throughput and the resolution required for measurement. A larger slit width allows for more light throughput. However, more light throughput results in poorer resolution. When choosing a slit width at which to operate the monochromator, both the input and output ports must be set to the same slit width. (See Figure 4.) Focused light enters the monochromator through the entrance slit, and is redirected by the collimating mirror toward the grating. The grating directs the light toward the focusing mirror, which then redirects the chosen wavelength toward the exit slit. At the exit slits, quasi-monochromatic light is emitted4.

A fixed width slit being installed into an Oriel Cornerstone 130 monochromator.

Figure 4. A fixed width slit being installed into an Oriel Cornerstone 130 monochromator.Photo courtesy of Newport Corp.

Measuring quantum efficiencies

Measuring quantum efficiency (QE) over a range of different wavelengths to measure a device’s QE at each photon energy level is an ideal task for a tunable light source. The QE of a photoelectric material for photons with energy below the band gap is zero. The QE value of a light-sensing device such as a solar cell indicates the amount of current that the cell will produce when irradiated by photons of a particular wavelength. The principle of QE measurement is to count the proportion of carriers extracted from the material’s valence band to the number of photons impinging on the surface. To do this, it is necessary to shine a calibrated, tunable light on the cell, while simultaneously measuring the output current. The key to accurate measurement of the QE/internal photon to current efficiency is to accurately know how much scanning light is incident on the device under test and how much current is generated. Thus, measurement of light output with a NIST (National Institute of Standards and Technology) traceable calibrated detector is necessary prior to testing since illumination of an absolute optical power is required.

External quantum efficiency (EQE) is the ratio of the number of photons incident on a solar cell to the number of generated charge carriers. Internal quantum efficiency (IQE) also considers the internal efficiency — that is, the losses associated with the photons absorbed by nonactive layers of the cell. By comparison, EQE is much more straightforward to measure, and gives a direct parameter of how much output current will be contributed to the output circuit per incident photon at a given wavelength. IQE is a more in-depth parameter, taking into account the photoelectric efficiency of all composite layers of a material. In an IQE measurement, these losses from nonactive layers of the material are measured in order to calculate a net quantum efficiency — a much truer efficiency measurement.

Understanding the conversion efficiency as a function of the wavelength of light impingent on the cell makes QE measurement critical for materials research and solar cell design. With this data, the solar cell composition and topography can be modified to optimize conversion over the broadest possible range of wavelengths.

As a formula, it is given by IQE = EQE/(1 − R), where R is the reflectivity, direct and diffuse, of the solar cell. The IQE is an indication of the capacity of the active layers of the solar cell to make good use of the absorbed photons. It is always higher than the EQE, but should never exceed 100 percent, with the exception of multiple-exciton generation. Figure 5 illustrates how the tunable light source is used to illuminate the solar cell to perform an IQE measurement. The software controls all components of the measurement system, including the monochromator and data acquisition5.

A sample QE measurement system using the components of a tunable light source.

Figure 5. A sample QE measurement system using the components of a tunable light source. Photo courtesy of Newport Corp.

To measure quantum efficiency in 10-nm wavelength steps, the slit size of the monochromator is typically hundreds of micron in width. The slit width is reduced approximately half if 5-nm wavelength increments are desired. However, output power of the monochromator is reduced by more than 50 percent if the slit width is halved. Lowering optical power impacts QE measurement since a solar cell responds to this diminished optical power with low output current. This can result in a poor signal-to-noise ratio, making a QE measurement error more likely. The detection of low current requires very sensitive equipment with the ability to measure current down to the pico-ampere level. To make for an easier signal measurement, optical power is typically increased. A DC arc source is the better choice for QE measurements made in 5-nm increments or lower due to the lamp’s arc size resulting in better monochromator throughput. However, a QTH lamp is the better choice if greater than 0.1 percent light stability is required, with the trade-off of not being able to measure in as precise wavelength increments as if an arc lamp was used.

Balance between optical power and resolution is an important consideration as it impacts the quality of the QE measurement. The selection of lamp type and monochromator specifications are important considerations for TLS design. To be considered a suitable component for the majority of spectroscopic applications, high-output power and stability, long lifetime of the lamp, and broadband spectral emission with high resolution capability are required for the TLS.

Meet the authors

John Park, new product development manager at Newport Corp., has designed and developed numerous spectroscopy instruments for the photonics industry for over 10 years. He holds two granted patents and is a graduate from University of California, Irvine, with a Ph.D. in electrical engineering; email: john.park@newport.com. Jeff Eng is a product specialist for Oriel Spectroscopy Products at Newport Corp. His work experience includes application support, business-to-business sales and marketing activity of photonic light sources and detectors. He is a graduate of Rutgers University; email: jeff.eng@newport.com.

References

1. Newport Corp., Oriel Instruments TLS datasheet. Tunable Xe arch lamp sources.http://assets.newport.com/webDocuments-EN/images/39191.pdf.

2. Newport Corp., Oriel Instruments handbook: The Book of Photon Tools, light source section.

3. Newport Corp., Oriel Instruments OPS datasheet. OPS-A series arc lamp power supplies.http://assets.newport.com/webDocuments-EN/images/OPS-A%20Series%20Power%20Supply%20Datasheet.pdf.

4. J. M. Lerner and A. Thevenon (1988). The Optics of Spectroscopy. Edison, N.J.: Optical Systems/Instruments SA Inc.

5. K. Emery (2005). Handbook of Photovoltaic Science and Engineering, eds. A. Luque and S. Hegedus. Chapter 16: Measurement and characterization of solar cells and modules. Hoboken, N.J.: John Wiley & Sons Ltd.

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Targeting hematopoietic stem cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New technology uncovered the stem cell niche in the bone marrow

HSCs, Stem cells, hematopoiesis

Hematopoietic stem cells (HSCs) are so rare that it’s difficult to comprehensively localize dividing and non-dividing HSCs. Thus, there has controversy about their specific location in the bone marrow. A recent Nature publication reported that the HSCs resides mainly in perisinusoidal niches through out the bone marrow and there are no spatially distinct niches for dividing and non-dividing blood-forming stem cells. This group of researchers at UT Southwestern Medical Center started the generation of a GFP knock-in for the gene Ctnnal1, a generic marker for HSCs in mice (α-catulinGFP mice) and confirmed that α-catulin-GFP+c-kit+ cells represent blood-forming HSCs by showing that α-catulin-GFP+c-kit+ cells gave long term multi-lineage reconstitution of irradiated mice. Using a tissue-clearing technique and deep confocal imaging, they were able to image thousands of α-catulin-GFP+c-kit+ cells and see their relation to other cells. This publication improved the understanding of the microenvironment of HSCs in the bone marrow, which would significantly improve the safety and effectiveness of bone marrow transplantation.

Melih Acar, etc. (October 2015) Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature

 

Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal

AcarKS. KocherlakotaMM. MurphyJG. PeyerH OguroCN. InraC JaiyeolaZ ZhaoK Luby-Phelps & Sean J. Morrison
Nature526,126–130(01 October 2015)
   
       doi:10.1038/nature15250

 

Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial1. HSCs are rare and few can be found in thin tissue sections2, 3 or upon live imaging4, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as αcatulinGFP), we discover that αcatulinGFP is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of αcatulin−GFP+c-kit+ cells give long-term multilineage reconstitution of irradiated mice, indicating thatαcatulin−GFP+c-kit+ cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of αcatulin–GFP+c-kit+ cells and to digitally reconstruct large segments of bone marrow. The distribution of αcatulin–GFP+c-kit+ cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr+) and Cxcl12high niche cells, and approximately 85% of HSCs were within 10 μm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67+) and non-dividing (Ki-67), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr+Cxcl12high cells throughout the bone marrow.

 

Figure 1: Deep imaging of αcatulin−GFP+ HSCs in digitally reconstructed bone marrow.close

 

Deep imaging of [agr]-catulin-GFP+ HSCs in digitally reconstructed bone marrow.

a, Only 0.021 ± 0.006% of αcatulinGFP/+ bone marrow cells were GFP+ (n = 14 mice in 11 independent experiments). b, Nearly allαcatulin−GFP+c-kit+ bone marrow cells were CD150+CD48 (n = 9 mice in 3 independent experiments;

 

Extended Data Figure 3: αcatulin−GFP expression among haematopoietic cells is highly restricted to HSCs.

 

[agr]-catulin-GFP expression among haematopoietic cells is highly restricted to HSCs.

 

a, The frequency of αcatulin−GFP+ bone marrow cells in negative control αcatulin+/+ (WT) mice and α-catulinGFP/+ mice (n = 14 mice per genotype in 11 independent experiments). In all cases in this figure, percentages refer to the frequency of each population as a percentage of WBM cells. b, αcatulin−GFP+c-kit+ cells from Fig. 1b are shown (blue dots) along with all other bone marrow cells in the same sample (red dots). c, CD150+CD48LSK HSCs express αcatulin−GFP but CD150CD48LSK MPPs do not (n = 17 mice in 12 independent experiments). A minority of the αcatulin−GFP+c-kit+ cells had high forward scatter, lacked reconstituting potential, and were gated out when isolating HSCs by flow cytometry and when identifying HSCs during imaging (see Extended Data Fig. 5for further explanation). d, Linc-kitlowSca-1lowCD127+CD135+ common lymphoid progenitors (CLPs), Linc-kit+Sca-1CD34+CD16/32 common myeloid progenitors (CMPs), Linc-kit+Sca-1CD34+CD16/32+ granulocyte-macrophage progenitors (GMPs), and Linc-kit+Sca-1CD34CD16/32 megakaryocyte-erythroid progenitors (MEPs) did not express αcatulin−GFP. αcatulinGFP/+ and control cell populations had similar levels of background GFP signals that accounted for fewer than 1% of the cells in each population (n = 9 mice per genotype in 2 independent experiments).

 

Extended Data Figure 7: HSC density is higher in the diaphysis as compared to the metaphysis.

HSC density is higher in the diaphysis as compared to the metaphysis.

a, Schematic of a femur showing the separation of epiphysis/metaphysis from diaphysis. We divided metaphysis from diaphysis at the point where the central sinus branched (see red line in panels a, f,and i). This is also the point at wh…

 

 

Extended Data Figure 9: Bone marrow blood vessel types can be distinguished based on vessel diameter, continuity of basal lamina, morphology, and position; and no difference in the distribution of HSCs in the bone marrow of male and female mice was detected.close

Bone marrow blood vessel types can be distinguished based on vessel diameter, continuity of basal lamina, morphology, and position; and no difference in the distribution of HSCs in the bone marrow of male and female mice was detected.

a, b, Schematic (a) and properties (b) of blood vessels in the bone marrow. Blood enters the marrow through arterioles that branch as they become smaller in diameter and approach the endosteum, where they connect to smaller diameter tra…

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Reverse Engineering of Vision

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

CMU announces research project to reverse-engineer brain algorithms, funded by IARPA

A Human Genome Project-level plan to make computers learn like humans
February 5, 2016   http://www.kurzweilai.net/cmu-announces-research-project-to-reverse-engineer-brain-algorithms-funded-by-iarpa

http://www.kurzweilai.net/images/neural-network-CMU.jpg

Individual brain cells within a neural network are highlighted in this image obtained using a fluorescent imaging technique (credit: Sandra Kuhlman/CMU)

Carnegie Mellon University is embarking on a five-year, $12 million research effort to reverse-engineer the brain and “make computers think more like humans,” funded by the U.S. Intelligence Advanced Research Projects Activity (IARPA). The research is led by Tai Sing Lee, a professor in the Computer Science Department and the Center for the Neural Basis of Cognition (CNBC).

The research effort, through IARPA’s Machine Intelligence from Cortical Networks (MICrONS) research program, is part of the U.S. BRAIN Initiative to revolutionize the understanding of the human brain.

A “Human Genome Project” for the brain’s visual system

“MICrONS is similar in design and scope to the Human Genome Project, which first sequenced and mapped all human genes,” Lee said. “Its impact will likely be long-lasting and promises to be a game changer in neuroscience and artificial intelligence.”

The researchers will attempt to discover the principles and rules the brain’s visual system uses to process information. They believe this deeper understanding could serve as a springboard to revolutionize machine learning algorithms and computer vision.

In particular, the researchers seek to improve the performance of artificial neural networks — computational models for artificial intelligence inspired by the central nervous systems of animals. Interest in neural nets has recently undergone a resurgence thanks to growing computational power and datasets. Neural nets now are used in a wide variety of applications in which computers can learn to recognize faces, understand speech and handwriting, make decisions for self-driving cars, perform automated trading and detect financial fraud.

How neurons in one region of the visual cortex behave

“But today’s neural nets use algorithms that were essentially developed in the early 1980s,” Lee said. “Powerful as they are, they still aren’t nearly as efficient or powerful as those used by the human brain. For instance, to learn to recognize an object, a computer might need to be shown thousands of labeled examples and taught in a supervised manner, while a person would require only a handful and might not need supervision.”

To better understand the brain’s connections, Sandra Kuhlman, assistant professor of biological sciences at Carnegie Mellon and the CNBC, will use a technique called “two-photon calcium imaging microscopy” to record signaling of tens of thousands of individual neurons in mice as they process visual information, an unprecedented feat. In the past, only a single neuron, or tens of neurons, typically have been sampled in an experiment, she noted.

“By incorporating molecular sensors to monitor neural activity in combination with sophisticated optical methods, it is now possible to simultaneously track the neural dynamics of most, if not all, of the neurons within a brain region,” Kuhlman said. “As a result we will produce a massive dataset that will give us a detailed picture of how neurons in one region of the visual cortex behave.”

A multi-institution research team

Other collaborators are Alan Yuille, the Bloomberg Distinguished Professor of Cognitive Science and Computer Science at Johns Hopkins University, and another MICrONS team at the Wyss Institute for Biologically Inspired Engineering, led by George Church, professor of genetics at Harvard Medical School.

The Harvard-led team, working with investigators at Cold Spring Harbor Laboratory, MIT, and Columbia University, is developing revolutionary techniques to reconstruct the complete circuitry of the neurons recorded at CMU. The database, along with two other databases contributed by other MICrONS teams, unprecedented in scale, will be made publicly available for research groups all over the world.

In this MICrONS project, CMU researchers and their collaborators in other universities will use these massive databases to evaluate a number of computational and learning models as they improve their understanding of the brain’s computational principles and reverse-engineer the data to build better computer algorithms for learning and pattern recognition.

“The hope is that this knowledge will lead to the development of a new generation of machine learning algorithms that will allow AI machines to learn without supervision and from a few examples, which are hallmarks of human intelligence,” Lee said.

The CNBC is a collaborative center between Carnegie Mellon and the University of Pittsburgh. BrainHub is a neuroscience research initiative that brings together the university’s strengths in biology, computer science, psychology, statistics and engineering to foster research on understanding how the structure and activity of the brain give rise to complex behaviors.

The MICrONS team at CMU allso includes Abhinav Gupta, assistant professor of robotics; Gary Miller, professor of computer science; Rob Kass, professor of statistics and machine learning and interim co-director of the CNBC; Byron Yu, associate professor of electrical and computer engineering and biomedical engineering and the CNBC; Steve Chase, assistant professor of biomedical engineering and the CNBC; and Ruslan Salakhutdinov, one of the co-creators of the deep belief network, a new model of machine learning that was inspired by recurrent connections in the brain, who will join CMU as an assistant professor of machine learning in the fall.

Other members of the team include Brent Doiron, associate professor of mathematics at Pitt, and Spencer Smith, assistant professor of neuroscience and neuro-engineering at the University of North Carolina.

Not all machine-intelligence experts are on board with reverse-engineering the brain. In a Facebook post today, Yann LeCun, Director of AI Research at Facebook and a professor at New York University, asked the question in a recent lecture, “Should we copy the brain to build intelligent machines?” “My answer was ‘no, because we need to understand the underlying principles of intelligence to know what to copy. But we should draw inspiration from biology.’”

 

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