Transitioning Clinical Trial Paper Source Records to the Digital World

 

We would appreciate any thoughts on the following.

 

Back in the “old days,“ data collected from paper source records were transcribed to paper case report forms, which were then “mailed“ to the pharma company to perform data entry. When EDC systems were established, the pharmaceutical industry transferred the transcription of these data, by the sites, to the EDC systems. As a result, with the concept of the need to perform 100% source document verification (SDV), the workload for both the sites and CRAs did not change. What did change was that data entry into electronic databases no longer was performed by pharmaceutical companies or CROs, but was now performed by the sites.

 

The whole idea behind efficiencies of electronic systems is interoperability. Nevertheless, there is a new phenomenon on the horizon which is perhaps being perceived as an advantage by clinical research sites. The sites are now being being told that they can create their own electronic source documentation, not dissimilar from creating a paper source record. The big question is not about the validation of these systems, which of course could be an issue, but how these data are mapped and eventually transferred to the study database. We were told recently, that sites are re-entering these electronically collected source data into EDC systems by opening 2 screens, and then entering the data a second time. Are we going to end up with just another version of SDV? Imagine having to deal with transferring these data from multiple site source systems into one or more EDC systems. This challenge is also not different where source data are collected within multiple EMR systems.

 

There are several alternative solutions to support the paperless clinical trial by eliminating or reducing paper source records generated by the sites. Target Health has developed and implemented a patented bring-your-own-device (BYOD) web-based system, that allows for direct data at the time of the office visit, with the simultaneous creation of  electronic source documents. This user friendly system allows for the generation of read-only files maintained under the control of the sites, prior to the data being transferred to the EDC database. There now both FDA and EMA approval of products developed using our system. Other systems include dedicated tablets that are distributed to the clinical sites that also allow for direct data entry at the time of the patient encounter. In addition, real-time transfer of data from multiple EMR systems into one EDC data base is in the very near future, as well as direct capture and integration of data coming from mobile devices.

 

Springtime in NYC

Finally Spring has arrived in NYC. ©Target Health Inc. April 2018

 

For more information about Target Health contact Warren Pearlson (212-681-2100 ext. 165). For additional information about software tools for paperless clinical trials, please also feel free to contact Dr. Jules T. Mitchel. The Target Health software tools are designed to partner with both CROs and Sponsors. Please visit the Target Health Website.

 

Joyce Hays, Founder and Editor in Chief of On Target

Jules Mitchel, Editor

 

QUIZ

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What Are Genome Editing and CRISPR-CAS9?

This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license. Wikipedia

 

Genome editing (also called gene editing) is a group of technologies that give scientists the ability to change an organism’s 1) ____. These technologies allow genetic material to be added, removed, or altered at particular locations in the 2) ____. Several approaches to genome editing have been developed. A recent one is known as CRISPR-Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9. The CRISPR-Cas9 system has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate, and more efficient than other existing genome editing methods.

 

CRISPR-Cas9 was adapted from a naturally occurring genome editing system in 3) ____. The bacteria capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR 4) ____. The CRISPR arrays allow the bacteria to “remember“ the viruses (or closely related ones). If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays to target the viruses’ DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus.

 

The CRISPR-Cas9 system works similarly in the lab. Researchers create a small piece of RNA with a short “guide“ sequence that attaches (binds) to a specific target sequence of DNA in a genome. The RNA also binds to the Cas9 enzyme. As in bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location. Although Cas9 is the enzyme that is used most often, other enzymes (for example Cpf1) can also be used. Once the DNA is cut, researchers use the cell’s own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA 5) ____.

 

Genome editing is of great interest in the prevention and treatment of human diseases. Currently, most research on genome editing is done to understand diseases using cells and animal models. Scientists are still working to determine whether this approach is safe and effective for use in people. It is being explored in research on a wide variety of diseases, including single-gene disorders such as 6) ____ ____, hemophilia and 7) ____ cell disease. It also holds promise for the treatment and prevention of more complex diseases, such as cancer, heart disease, mental illness, and human immunodeficiency virus (HIV) infection.

 

Ethical concerns arise when genome editing, using technologies such as CRISPR-Cas9, is used to alter human genomes. Most of the changes introduced with genome editing are limited to somatic cells, which are cells other than egg and sperm cells. These changes affect only certain tissues and are not passed from one generation to the next. However, changes made to genes in egg or sperm cells (8) ____ cells) or in the genes of an embryo could be passed to future generations. Germline cell and embryo genome editing bring up a number of 9) ____ challenges, including whether it would be permissible to use this technology to enhance normal human traits (such as height or intelligence). Based on concerns about ethics and safety, germline cell and embryo genome editing are currently 10) ____ in many countries. Source: NIH.gov

 

ANSWERS: 1) DNA; 2) genome; 3) bacteria; 4) arrays; 5) sequence; 6) cystic fibrosis; 7) sickle; 8) germline; 9) ethical; 10) illegal

 

CRISPR

Double Strand DNA Breaks Introduced by CRISPR-Cas9 Allows Further Genetic Manipulation By Exploiting Endogenous DNA Repair Mechanisms.

Graphic credit: by Guido4 – Own work; CC BY-SA 4.0; File:16 Hegasy DNA Rep Wiki E CCBYSA.png; Created: 1 November 2017; Wikipedia Creative Commons

 

The discovery of clustered DNA repeats occurred independently in three parts of the world. The first description of what would later be called CRISPR is from Osaka University researcher Yoshizumi Ishino and his colleagues in 1987. They accidentally cloned part of a CRISPR together with the iap gene, the target of interest. The organization of the repeats was unusual because repeated sequences are typically arranged consecutively along DNA. They studied the relation of “iap“ to the bacterium E. coli. The function of the interrupted clustered repeats was not known at the time. In 1993 researchers of Mycobacterium tuberculosis in the Netherlands published two articles about a cluster of interrupted direct repeats (DR) in this bacterium. These researchers recognized the diversity of the DR-intervening sequences among different strains of M. tuberculosis and used this property to design a typing method that was named spoligotyping, which is still in use today. At the same time, repeats were observed in the archaeal organisms of Haloferax and Haloarcula species, and their function was studied by Francisco Mojica at the University of Alicante in Spain. Although his hypothesis turned out to be wrong, Mojica’s supervisor surmised at the time that the clustered repeats had a role in correctly segregating replicated DNA into daughter cells during cell division because plasmids and chromosomes with identical repeat arrays could not coexist in Haloferax volcanii. Transcription of the interrupted repeats was also noted for the first time. By 2000, Mojica performed a survey of scientific literature and one of his students a search in published genomes with a program devised by himself. They found interrupted repeats in 20 species of microbes, and it was the first time different repeats with the same properties were identified as belonging to the same family, not yet known as CRISPR. In 2001, Mojica and Ruud Jansen, who was searching for additional interrupted repeats, proposed the acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to alleviate the confusion stemming from the numerous acronyms used to describe the sequences in the scientific literature.

 

CRISPR-Associated Systems

 

A major addition to the understanding of CRISPR came with Jansen’s observation that the prokaryote repeat cluster was accompanied by a set of homologous genes that make up CRISPR-associated systems or cas genes. Four cas genes (cas 1 – 4) were initially recognized. The Cas proteins showed helicase and nuclease motifs, suggesting a role in the dynamic structure of the CRISPR loci. In this publication the acronym CRISPR was coined as the universal name of this pattern. However, the CRISPR function remained enigmatic.

 

Simplified diagram of a CRISPR locus. The three major components of a CRISPR locus are shown: cas genes, a leader sequence, and a repeat-spacer array. Repeats are shown as gray boxes and spacers are colored bars. The arrangement of the three components is not always as shown. In addition, several CRISPRs with similar sequences can be present in a single genome, only one of which is associated with cas genes. In 2005, three independent research groups showed that some CRISPR spacers are derived from phage DNA and extrachromosomal DNA such as plasmids. In effect, the spacers are fragments of DNA gathered from viruses that previously tried to attack the cell. The source of the spacers was a sign that the CRISPR/cas system could have a role in adaptive immunity in bacteria. All three studies proposing this idea were initially rejected by high-profile journals, but eventually appeared in other journals. The first publication proposing a role of CRISPR-Cas in microbial immunity, by the researchers at the University of Alicante, predicted a role for the RNA transcript of spacers on target recognition in a mechanism that could be analogous to the RNA interference system used by eukaryotic cells. This hypothesis had already been defended in a pre-doc examination and one scientific meeting in 2004. Koonin and colleagues extended this RNA interference hypothesis by proposing mechanisms of action for the different CRISPR-Cas subtypes according to the predicted function of their proteins.

 

Experimental work by several groups revealed the basic mechanisms of CRISPR-Cas immunity. In 2007 the first experimental evidence that CRISPR was an adaptive immune system was published. A CRISPR region in Streptococcus thermophilus acquired spacers from the DNA of an infecting bacteriophage. The researchers manipulated the resistance of S. thermophilus to phage by adding and deleting spacers whose sequence matched those found in the tested phages. In 2008, Brouns and Van der Oost identified a complex of Cas proteins (called Cascade) that in E. coli cut the CRISPR RNA precursor within the repeats into mature spacer-containing RNA molecules (crRNA), which remained bound to the protein complex. Moreover, it was found that Cascade, crRNA and an helicase/nuclease (Cas3) were required to provide a bacterial host with immunity against infection by a DNA virus. By designing an anti-virus CRISPR, they demonstrated that two orientations of the crRNA (sense/antisense) provided immunity, indicating that the crRNA guides were targeting dsDNA. That year Marraffini and Sontheimer indeed confirmed that a CRISPR sequence of S. epidermidis targeted DNA and not RNA to prevent conjugation. This finding was at odds with the proposed RNA-interference-like mechanism of CRISPR-Cas immunity, although a CRISPR-Cas system that targets foreign RNA was later found in Pyrococcus furiosus. A 2010 study showed that CRISPR-Cas cuts both strands of phage and plasmid DNA in S. thermophilus.

 

Cas9

 

Researchers studied a simpler CRISPR system from Streptococcus pyogenes that relies on the protein Cas9. The Cas9 endonuclease is a four-component system that includes two small RNA molecules named CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). Jennifer Doudna and Emmanuelle Charpentier re-engineered the Cas9 endonuclease into a more manageable two-component system by fusing the two RNA molecules into a “single-guide RNA“ that, when combined with Cas9, could find and cut the DNA target specified by the guide RNA. By manipulating the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target any DNA sequence for cleavage. Another group of collaborators comprising Siksnys together with Gasiunas, Barrangou and Horvath showed that Cas9 from the S. thermophilus CRISPR system can also be reprogrammed to target a site of their choosing by changing the sequence of its crRNA. These advances fueled efforts to edit genomes with the modified CRISPR-Cas9 system. Feng Zhang’s and George Church’s groups simultaneously described genome editing in human cell cultures using CRISPR-Cas9 for the first time. It has since been used in a wide range of organisms, including baker’s yeast (Saccharomyces cerevisiae), the opportunistic pathogen C. albicans, zebrafish (D. rerio), fruit flies (Drosophila melanogaster), nematodes (C. elegans), plants, mice, monkeys and human embryos.

 

CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes. The CRIPSR/Cas9 system has shown to make effective gene edits in Human tripronuclear zygotes first described in a 2015 paper by Chinese scientists P. Liang and Y. Xu. The system made a successful cleavage of mutant Beta-Hemoglobin (HBB) in 28 out of 54 embryos. 4 out of the 28 embryos were successfully recombined using a donor template given by the scientists. The scientists showed that during DNA recombination of the cleaved strand, the homologous endogenous sequence HBD competes with the exogenous donor template. DNA repair in human embryos is much more complicated and particular than in derived stem cells.

 

Cpf1

 

In 2015, the nuclease Cpf1 was discovered in the CRISPR/Cpf1 system of the bacterium Francisella novicida. Cpf1 showed several key differences from Cas9 including: causing a ?staggered’ cut in double stranded DNA as opposed to the ?blunt’ cut produced by Cas9, relying on a ?T rich’ PAM (providing alternate targeting sites to Cas9) and requiring only a CRISPR RNA (crRNA) for successful targeting. By contrast Cas9 requires both crRNA and a transactivating crRNA (tracrRNA). These differences may give Cpf1 some advantages over Cas9. For example, Cpf1’s small crRNAs are ideal for multiplexed genome editing, as more of them can be packaged in one vector than can Cas9’s sgRNAs. As well, the sticky 5′ overhangs left by Cpf1 can be used for DNA assembly that is much more target-specific than traditional Restriction Enzyme cloning. Finally, Cpf1 cleaves DNA 18-23 bp downstream from the PAM site. This means there is no disruption to the recognition sequence after repair, and so Cpf1 enables multiple rounds of DNA cleavage. By contrast, since Cas9 cuts only 3 bp upstream of the PAM site, the NHEJ pathway results in indel mutations which destroy the recognition sequence, thereby preventing further rounds of cutting. In theory, repeated rounds of DNA cleavage should cause an increased opportunity for the desired genomic editing to occur.

 

Watching the Brain’s Lining Heal After Head Injury

 

Following head injury, the protective lining that surrounds the brain may get a little help from its friends: immune cells that spring into action to assist with repairs. According to a study published online in Nature Immunology (16 April 2018), scientists from the National Institutes of Health watched in real-time as different immune cells in the mouse took on carefully timed jobs to fix the damaged lining of the brain, also known as meninges. The meninges are a collection of membranes that line the central nervous system and help protect brain and spinal cord tissue from various forms of injury. Damage to the meninges can cause cell death in underlying brain tissue. These results may help provide clues to the discovery that the meninges in humans may heal following mild traumatic brain injury (mTBI) and why additional hits to the head can be so devastating. The study came about from an observation on MRI scans of adult patients who experienced a concussion or mTBI. Around half of patients with mTBI show evidence of injury to blood vessels in the meninges, which appears on MRI scans as a vascular dye leaking out of the damaged vessels. The authors found that while most patients had repaired their leaky blood vessels within 20 days, 17% of patients still showed leakage on their MRI scans three months after injury, indicating ongoing meningeal damage and incomplete recovery.

 

To learn more about the recovery process, the authors used state-of-the-art imaging tools to watch, in real-time, what happened in the mouse meninges up to one week after injury. They also developed a method of analyzing where immune cells gathered in the damaged meninges during the repair process. Results showed that within the first day of injury, immune cells from the blood called inflammatory monocytes entered the core of the injured meningeal tissue and started clearing away dead cells. These cells were assisted a few days later by a different type of blood monocyte that worked around the lesion edge to help rebuild damaged blood vessels, which were completely restored and fully functional within a week. It was observed that the actions of these different immune cell types did not overlap and that blocking the activity of one did not cause the other to take over.

 

The authors also found that the timing of a second head injury has a significant impact on the repair process in mice. A second injury experienced within one day of the first TBI led to additional inflammation and the wound healing phase of repair, during which blood vessels are fixed, did not occur. However, if the re-injury occurred after a few days, once the wound healing phase had already begun, there was no effect on the meningeal repair process and blood vessels were rebuilt normally. According to the authors, the timing of a second head injury may determine whether the meninges can be repaired, since it has been shown on a cellular level, that two or more head injuries within a very short amount of time can have really dire consequences for the brain lining and its ability to repair. Thus it is possible that patients who did not fully recover following a head injury may have had problems with the first phase of the repair process.

 

Experiments revealed that the molecule matrix metalloproteinase 2 (Mmp2) may play a critical role in the restoration of blood vessels. The wound-healing immune cells release Mmp2, which breaks down the matrix, or glue, holding cells together, allowing room for blood vessels to be rebuilt. When Mmp2 was blocked, there was a large decrease in the number of vessels that were repaired. According to the authors, further research is needed to uncover additional molecules and genes involved in the repair processes and identify ways to speed up the course of recovery following head injury.

 

Human Derived Antibody Prevents Malaria in Mice

 

In the latest World Malaria Report of the World Health Organization, there were 216 million cases of malaria worldwide in 2016 resulting in an estimated 445,000 deaths. Almost every malarial death is caused by Plasmodium falciparum, and 91% of death occurs in Africa. Children under five years of age are most affected, accounting for two-third of the total deaths. Currently, there is no highly effective, long-lasting vaccine to prevent malaria.

 

P. falciparum is a unicellular protozoan parasite of humans, and the deadliest species of Plasmodium that cause malaria in humans. It is transmitted through the bite of a female Anopheles mosquito and is responsible for roughly 50% of all malaria cases. It causes the disease’s most dangerous form called falciparum malaria. It is also associated with the development of blood cancer (Burkitt’s lymphoma) and is classified as Group 2A carcinogen.

 

The human parasite originated from the malarial parasite Laverania found in gorillas, around 10,000 years ago. Alphonse Laveran was the first to identify the parasite in 1880, and named it Oscillaria malariae. Ronald Ross discovered its transmission by mosquito in 1897. Giovanni Battista Grassi elucidated the complete transmission from a female anopheline mosquito to humans in 1898. In 1897, William H. Welch created the name Plasmodium falciparum, which ICZN formally adopted in 1954.

 

According to an article published in Nature Medicine (Volume 24: 408-416, 2018), a human antibody has been discovered that protected mice from infection with P. falciparum. The research findings now provide the basis for future testing in humans to determine if the antibody can provide short-term protection against malaria, and also may aid in vaccine design. For the study, the authors, isolated the antibody, called CIS43, from the blood of a volunteer who had received an experimental vaccine made from whole, weakened malaria parasites (PfSPZ Vaccine-Sanaria). The volunteer was later exposed to infectious malaria-carrying mosquitoes under carefully controlled conditions and did not become infected.

 

In followup experiments, in two different models of malaria infection in mice, CIS43 was highly effective at preventing malaria infection. If confirmed through additional studies in people, CIS43 could be developed as a prophylactic measure to prevent infection for several months after administration. Such a prophylactic antibody could be useful for tourists, health care workers, military personnel or others who travel to areas where malaria is common. Moreover, if the antibody prevented malaria infection for up to six months, it might be combined with antimalarial drugs and be deployed as part of mass drug administration efforts that potentially could eliminate the disease in malaria-endemic regions.

 

Detailed examination of CIS43 revealed that it works by binding to a specific portion (epitope) of a key parasite surface protein. This epitope occurs only once along the length of the surface protein. In addition, the CIS43-binding epitope is conserved across 99.8% of all known strains of P. falciparum, making it an attractive target for next-generation experimental malaria vaccines designed to elicit production of this neutralizing antibody. The authors are planning to assess the safety and protective efficacy of the newly described CIS43 antibody next year in controlled human malaria infection challenge trials.

 

Digital Health Guidance From FDA

 

The FDA has recognized that it can help encourage digital health innovation by making its policies and processes more efficient and modernizing its regulatory tools. As part of the FDA’s Digital Health Innovation Action Plan issued in 2017, the agency committed to developing a new regulatory framework for reviewing software as a medical device and issuing a suite of guidances to provide transparency and clarity to product developers about the agency’s regulatory process.

 

On April 26, 2018, the FDA published an important progress update on the software precertification pilot program, the FDA’s proposed voluntary pathway for pre-certifying companies to enable a more streamlined review of their software as a medical device. The FDA is releasing a working model for its vision of the pilot, which outlines the most critical components of the pilot, like the precertification of companies, the premarket review process and postmarket surveillance. The agency is asking for public comment on the working model in order to obtain the critical feedback necessary to continue developing the program.

 

In addition to the pilot progress update, the FDA is releasing a draft guidance that addresses an important provision of the 21st Century Cures Act. The draft guidance offers additional clarity about where the FDA sees its role in digital health, and importantly, where the agency will not be involved. Since medical products may contain several functions, some of which are subject to the FDA’s regulatory oversight as medical devices, while others, in isolation, are not, the draft guidance, entitled, “Multiple Function Device Products: Policy and Considerations,“ explains the FDA’s proposed regulatory approach and policy for all multiple function device products. This guidance clarifies when and how the agency intends to look at the impact of non-regulated functions on the safety and effectiveness of device functions that are subject to FDA review. For example, consider a hospital monitor that detects and transmits vital patient signal information like heart rhythms into a patient’s electronic health record. In this case, the FDA would only review the heart monitor function, unless the transmission function impacts the safety or effectiveness of the monitor function or adversely affects the monitor capability. In that case, the developer only needs to show that they’ve addressed any potential for an adverse impact between the two different functions – the one the FDA oversees, and the one it does not. This example and others provided in the guidance demonstrate that by providing greater transparency and clarity on how the agency intends to approach these multiple function products, the FDA hopes to further encourage innovation in this important field.

 

Refreshing Leafy Salad with Melon, Pistachios, Cheese, Cranberries and a Light Citrus Garlic Dressing

A Perfect Summer Salad!  ©Joyce Hays, Target Health Inc.

 

Salad Ingredients

 

2 cups, assorted baby lettuce, shredded, approximately 5-6 ounces

2 cups, baby spinach, shredded

2 small to medium cucumbers, peeled

1/2 cup very fresh mozzarella, cubed

1/3 cup dried cranberries

1/3 cup pistachios

1/4 of a melon (you decide), cut bite-size

 

Dressing Ingredients

2 Tablespoons grapeseed oil

1 or 2 fresh garlic cloves, squeezed

Juice from 1/2 of an Orange

Juice from 1/2 of a Lemon

Pinch salt

Pinch black pepper

Pinch chili flakes

 

Fresh summer ingredients.  ©Joyce Hays, Target Health Inc.

 

Ingredients getting cut up. Notice how little dressing is needed. ©Joyce Hays, Target Health Inc.

 

Make enough; this salad will go fast!  ©Joyce Hays, Target Health Inc.

 

This meal was a delicious low calorie comfort food fest with some of our favorite dishes. First we clinked our glasses of chilled Vice chardonnay, just because we were enjoying being lazy, this weekend.

 

The recipe above was the first course and a delicious change from our usual tomato/avocado salad. Then we had eggplant parmesan, a recipe that was shared on this newsletter, long ago. Easy to prepare, simple but good, baked garlic-y mini red potatoes. For dessert, home-made black bean brownies with cool whip on top.

 

A treat for New Yorkers was not only a second Tosca at the MetOpera, but the amazingly beautiful voice of soprano Anna Netrebko, singing with her husband, Azerbaijani tenor Yusif Eyvazov.

 

Below is Netrebko singing one of the most sublime arias ever written.  Each time I hear it, I tear up. I’ve heard all the great sopranos sing Vissi d’Arte and IMO, this is the best recording.

 

https://www.youtube.com/watch?v=fnMMXfr8FZc

Anna Netrebko – Vissi d’arte (Tosca, Act 2) by Giocomo Puccini

 

This long weekend was good for us and we hope for you also.

 

Icy Stag’s Leap Sauvignon Blanc   ©Joyce Hays, Target Health Inc.

 

From Our Table to Yours!

Bon Appetit!

 

Dartmouth engineers and physicians develop innovative stereovision system

Date:
April 26, 2018

Source:
Dartmouth College

Summary:
Researchers develop a new way to make back surgery safer, faster and more cost effective. Scientists have developed and tested a 3-dimensional, real-time optical tracking system, like a ‘Google Maps’ for the body.

 

Researchers at Dartmouth College have found a way to make back surgery safer, faster and more cost effective.

MRIs and CT scans help surgeons identify spine problems, like compressed vertebrae or herniated disks, but finding a clear path to those problem areas is not always as straightforward. Tissue and bone not only stand in the way, they can also move during spinal surgery, rendering a CT scan taken prior to surgery much less accurate.

To solve this problem, Dartmouth professors from the Thayer School of Engineering and the Geisel School of Medicine developed a 3-dimensional, real-time optical tracking system to guide back surgeons as they operate, like a Google Maps for the body, according to findings published in the journal Operative Neurosurgery.

Using a complex software algorithm and two cameras attached to a surgical microscope, the system produces real-time 3-dimensional digitized images on a monitor, according to the study. This type of tracked, calibrated stereoscopic camera system has been extensively used in brain surgery but until now has been unexplored for use in spinal surgery.

The surgeon can use this new intraoperative stereovision system (iSV) without any additional radiation or labor-intensive marking of key areas on the patient’s spine, to match up or co-register with the pre-operative CT scan, as some surgeons do today. This new mapping provides more accurate renderings of where spinal implants or other surgical tools and devices need to go during the procedure, and is expected to save up to 30 minutes, according to one of the study’s authors, Keith D. Paulsen, PhD, Robert A. Pritzker Professor of Biomedical Engineering at Thayer School of Engineering at Dartmouth.

Paulsen and the multidisciplinary team at Dartmouth’s Center for Surgical Innovation tested the new iSV system for accuracy and efficiency while operating on pig spines. Since completing this study, the team has taken its complex system one step further by converting it into a handheld “wand” that the surgeon can pass over the surgical area.

“By rendering images real-time, with a simple handheld tool, we believe we can make surgeries safer and less costly in the future,” said Paulsen.

Next up is fine-tuning the system and testing in humans. The National Institutes of Health has provided the Dartmouth team with another round of funding to continue testing. It could be several years before the system becomes widely available for human spinal surgeries.

Story Source:

Materials provided by Dartmouth CollegeNote: Content may be edited for style and length.


Journal Reference:

  1. Linton Evans, Jonathan D Olson, Yunliang Cai, Xiaoyao Fan, Keith D Paulsen, David W Roberts, Songbai Ji, S Scott Lollis. Stereovision Co-Registration in Image-Guided Spinal Surgery: Accuracy Assessment Using Explanted Porcine SpinesOperative Neurosurgery, 2018; DOI: 10.1093/ons/opy023

 

Source: Dartmouth College. “New imaging system makes back surgery safer, faster and less expensive: Dartmouth engineers and physicians develop innovative stereovision system.” ScienceDaily. ScienceDaily, 26 April 2018. <www.sciencedaily.com/releases/2018/04/180426085518.htm>.

Cryo-electron microscopy images reveal details of protein, RNA binding and possible drug-target sites

Date:
April 25, 2018

Source:
University of California – Berkeley

Summary:
Telomerase, because of its role in cancer and aging, has long been a target of drug companies who want to block it to stop the uncontrolled cell growth characteristic of cancer, or boost it to create a fountain of youth. The structure of the enzyme complex has been a mystery, however, until now. Scientists have finally obtained a detailed picture of the architecture of the RNA-protein complex, a breakthrough for drug design.

 

Shown is the space-filling model of human telomerase holoenzyme structure determined by cryo-electron microscopy (cryo-EM). Telomerase catalyzes the synthesis of telomeric DNA (green) at the ends of chromosomes (blue) to compensate for the loss of telomeres during genome replication. The structure comprises two lobes with distinct functionalities: the catalytic core (purple) responsible for DNA synthesis and an H/ACA ribonucleoprotein lobe (cyan, teal and light green) important for telomerase biogenesis and localization to the Cajal body (red dots and lower left in closeup). Given that telomerase regulation is implicated in cancer and aging, this first architectural visualization of human telomerase represents an important breakthrough in the telomerase field and telomerase-based therapeutic design.
Credit: Janet Iwasa

 

 

More than 30 years ago, when University of California, Berkeley researchers discovered telomerase — an enzyme that lengthens chromosome ends and prevents them from fraying enough to kill a cell — speculation ran wild about its role in aging and cancer, setting off a full-court press to produce drugs to activate or block the enzyme.

While neither telomerase-based anti-aging drugs, touted as a “fountain of youth,” nor anticancer drugs have yet appeared, the publication today by UC Berkeley scientists of the first detailed picture of the molecular structure of human telomerase should jump-start that effort, allowing more targeted drug screens and intelligent design of new drugs.

“It has been a long time coming. It took a lot of persistence,” said Kathleen Collins, a UC Berkeley professor of molecular and cell biology who has worked on the enzyme for 26 years.

Collins and Eva Nogales, also a professor of molecular and cell biology, are the senior authors of a paper describing the 3-D molecular structure of the human telomerase enzyme published this week in the journal Nature.

One bottleneck has been obtaining pure samples of this complex molecule, which is composed of an RNA backbone decorated by six types of protein that move around as they add DNA to the ends of chromosomes. Labs around the world have debated whether the enzyme operates singly or as conjoined twins, and how and how many proteins decorate the RNA backbone.

Without consensus on these questions, it has proven difficult to design a drug to target the molecular machine and either destroy telomerase activity — which could stop a cancer that has boosted its telomerase levels — or restart telomerase, perhaps to prime the body for rapid cell division after a bone marrow transplant.

The newly revealed structure still lacks fine detail, but combined with knowledge of the gene sequence of human telomerase, it provides enough information to start thinking about potential targets for drugs, said first author Thi Hoang Duong “Kelly” Nguyen, a Miller Institute postdoctoral fellow at UC Berkeley.

“The best previous images of human telomerase had a resolution of only 30 Ångstroms; we were able to get about 7 to 8 Ångstroms resolution using cryoelectron microscopy,” Kelly said. “When I got to the point where I could see all the subunits — we had 11 protein subunits in total — it was a moment of, ‘Wow, wow, this is how they all fit together.'”

Nguyen, Collins and Nogales are actively working to improve the resolution to 3 or 4 Ångstroms — about the size of two carbon atoms — which is sufficient for drug design.

Telomerase tops up the telomeres

Telomeres were first detected at a molecular level in the late 1970s by Elizabeth Blackburn, then at UC Berkeley and now president emerita of the Salk Institute for Biological Studies in La Jolla, California. Working with the ciliated protozoan Tetrahymena, she and colleagues showed that the ends of the chromosomes are capped by repeating sequences of DNA. Armed with knowledge of telomere sequence, researchers then showed that telomeres in tissues of multicellular organisms grow shorter each time a cell divides. The telomeres protect the DNA strands from fraying and getting damaged at their ends, much like the plastic tip on the end of a shoelace. The fact that they drop off with each cell division is thought to protect us from cancer, when a cell is hijacked and proliferates continually.

In 1985 at UC Berkeley, Blackburn and then-graduate student Carol Greider discovered telomerase, an enzyme that adds DNA back to the ends of chromosomes, lengthening them and extending the lifespan of the cell. Blackburn, Greider and another colleague, Jack Szosak, shared the 2009 Nobel Prize in Physiology or Medicine for the discovery of telomerase.

Scientists have since found that, in humans and other multicellular organisms, telomerase is expressed only in the embryo, not in most adult cells. That means that most cells at birth have a predetermined ability to grow and divide, after which they die. Many scientists believe that depleted telomeres are a major cause of aging.

Collins has been trying to determine the structure of telomerase ever since the first human telomerase protein was discovered in 1997, and she and her colleagues have discovered and extensively characterized many of the proteins in the large enzyme, as well as the broken-up hairpin structure of the RNA backbone of telomerase. Yet how they all fit together was an enigma because of conflicting results from many different labs.

Nguyen was able to isolate the active enzyme and purify it much better than anyone had before, and employed a new, state-of-the-art cryoelectron microscope to determine the structure of the active telomerase unambiguously. Cryo-EM is a technique for determining molecular structures of compounds that cannot be crystallized and imaged with X-rays, and its developers won the 2017 Nobel Prize in Chemistry.

Once the UC Berkeley team had the structure, Nguyen said, it became clear why genetic mutations in some of the proteins in telomerase interfere with the enzyme to cause disease. In 1999, Collins discovered the first known human disease caused by a telomerase mutation: a mutation in a telomerase protein called dyskerin that is responsible for a rare disease called dyskeratosis congenita. Patients develop anemia as well as skin and gut problems, and most frequently die from bone marrow failure.

The reason, Collins says, is that there are two dyskerin molecules bound to the RNA backbone that have to not only reach out to the network of other proteins but also touch one another, and disease-causing mutations prevent these linkages, crippling the ability of the RNA backbone to survive in cells. Some children with dyskeratosis congenita have telomerase levels about 25 percent of normal and a lifespan of less than two decades. Those with half the normal level of telomerase typically reach a health crisis in mid-life.

Collins is ecstatic to finally have a definitive structure for telomerase and looks forward to learning more about the intricate assembly process of one of the most complex enzymes in the body: a polymerase as complicated as the ribosome, which reads RNA to produce proteins.

“I didn’t think it would be this complicated when I decided to study this molecule,” she said. “I became curious about how telomerase works in 1991, when I was just finishing as a grad student, and I was looking for a really simple polymerase system in which to understand nucleic acid interactions. And I thought, my god you can’t be simpler than this. That was super naive.”

Story Source:

Materials provided by University of California – Berkeley. Original written by Robert Sanders. Note: Content may be edited for style and length.


Journal Reference:

  1. Thi Hoang Duong Nguyen, Jane Tam, Robert A. Wu, Basil J. Greber, Daniel Toso, Eva Nogales, Kathleen Collins. Cryo-EM structure of substrate-bound human telomerase holoenzymeNature, 2018; DOI: 10.1038/s41586-018-0062-x

 

Source: University of California – Berkeley. “Long-sought structure of telomerase paves way for new drugs for aging, cancer: Cryo-electron microscopy images reveal details of protein, RNA binding and possible drug-target sites.” ScienceDaily. ScienceDaily, 25 April 2018. <www.sciencedaily.com/releases/2018/04/180425131846.htm>.

CMU process takes advantage of a common printing defect

Date:
April 24, 2018

Source:
Carnegie Mellon University

Summary:
Researchers have used an inexpensive 3-D printer to produce flat plastic items that, when heated, fold themselves into predetermined shapes, such as a rose, a boat or even a bunny. These self-folding plastic objects represent a first step toward products such as flat-pack furniture that assume their final shapes with the help of a heat gun.

 

A computer-controlled process developed at Carnegie Mellon University enables flat pieces of plastic produced in an inexpensive 3-D printer to assume predetermined 3-D shapes when heated.
Credit: Carnegie Mellon University

 

 

Researchers at Carnegie Mellon University have used an inexpensive 3-D printer to produce flat plastic items that, when heated, fold themselves into predetermined shapes, such as a rose, a boat or even a bunny.

Lining Yao, assistant professor in the Human-Computer Interaction Institute and director of the Morphing Matter Lab, said these self-folding plastic objects represent a first step toward products such as flat-pack furniture that assume their final shapes with the help of a heat gun. Emergency shelters also might be shipped flat and fold into shape under the warmth of the sun.

Self-folding materials are quicker and cheaper to produce than solid 3-D objects, making it possible to replace noncritical parts or produce prototypes using structures that approximate the solid objects. Molds for boat hulls and other fiberglass products might be inexpensively produced using these materials.

Yao will present her group’s research on this method, which she calls Thermorph, at CHI 2018, the Conference on Human Factors in Computing Systems, April 21-26 in Montreal, Canada.

Other researchers have explored self-folding materials, but typically have used exotic materials or depended on sophisticated processing techniques not widely available. Yao and her research team were able to create self-folding structure by using the least expensive type of 3-D printer — an FDM printer — and by taking advantage of warpage, a common problem with these printers.

“We wanted to see how self-assembly could be made more democratic — accessible to many users,” Yao said.

FDM printers work by laying down a continuous filament of melted thermoplastic. These materials contain residual stress and, as the material cools and the stress is relieved, the thermoplastic tends to contract. This can result in warped edges and surfaces.

“People hate warpage,” Yao said. “But we’ve taken this disadvantage and turned it to our advantage.”

To create self-folding objects, she and her team precisely control this process by varying the speed at which thermoplastic material is deposited and by combining warp-prone materials with rubber-like materials that resist contracture.

The objects emerge from the 3-D printer as flat, hard plastic. When the plastic is placed in water hot enough to turn it soft and rubbery — but not hot enough to melt it — the folding process is triggered.

Though they used a 3-D printer with standard hardware, the researchers replaced the machine’s open source software with their own code that automatically calculates the print speed and patterns necessary to achieve particular folding angles.

“The software is based on new curve-folding theory representing banding motions of curved area. The software based on this theory can compile any arbitrary 3-D mesh shape to an associated thermoplastic sheet in a few seconds without human intervention,” said Byoungkwon An, a research affiliate in HCII.

“It’s hard to imagine this being done manually,” Yao said.

Though these early examples are at a desktop scale, making larger self-folding objects appears feasible.

“We believe the general algorithm and existing material systems should enable us to eventually make large, strong self-folding objects, such as chairs, boats or even satellites,” said Jianzhe Gu, HCII research intern.

Video: https://vimeo.com/265829811

Story Source:

Materials provided by Carnegie Mellon UniversityNote: Content may be edited for style and length.


Journal Reference:

  1. Byoungkwon An, Hsiang-Yun Wu, Teng Zhang, Lining Yao, Ye Tao, Jianzhe Gu, Tingyu Cheng, Xiang ‘Anthony’ Chen, Xiaoxiao Zhang, Wei Zhao, Youngwook Do, Shigeo Takahashi. ThermorphConference on Human Factors in Computing Systems, 2018 DOI: 10.1145/3173574.3173834

 

Source: Carnegie Mellon University. “Cheap 3-D printer can produce self-folding materials: CMU process takes advantage of a common printing defect.” ScienceDaily. ScienceDaily, 24 April 2018. <www.sciencedaily.com/releases/2018/04/180424112836.htm>.

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