Stem Cell Clinic

DNA Nanomachines Are Opening Medicine to the World of Physics – Singularity Hub

When I imagine the inner workings of a robot, I think hard, cold mechanics running on physics: shafts, wheels, gears. Human bodies, in contrast, are more of a contained molecular soup operating on the principles of biochemistry.

Yet similar to robots, our cells are also attuned to mechanical forcesjust at a much smaller scale. Tiny pushes and pulls, for example, can urge stem cells to continue dividing, or nudge them into maturity to replace broken tissues. Chemistry isnt king when it comes to governing our bodies; physical forces are similarly powerful. The problem is how to tap into them.

In a new perspectives article in Science, Dr. Khalid Salaita and graduate student Aaron Blanchard from Emory University in Atlanta point to DNA as the solution. The team painted a futuristic picture of DNA mechanotechnology, in which we use DNA machines to control our biology. Rather than a toxic chemotherapy drip, for example, a cancer patient may one day be injected with DNA nanodevices that help their immune cells better grab ontoand snuff outcancerous ones.

For a long time, said Salaita, scientists have been good at making micro devices, hundreds of times smaller than the width of a human hair. Its been more challenging to make functional nano devices, thousands of times smaller than that. But using DNA as the component parts is making it possible to build extremely elaborate nano devices because the DNA parts self-assemble.

Just as the steam engine propelled civilization through the first industrial revolution, DNA devices may fundamentally change medicine, biological research, and the development of biomaterials, further merging man and machine.

When picturing a tiny, whirling machine surveying the body, DNA probably isnt the first candidate that comes to mind. Made up of long chains of four lettersA, T, C, and GDNA is normally secluded inside a tiny porous cage in every cell, in the shape of long chains wrapped around a protein core.

Yet several properties make DNA a fascinating substrate for making mechano-machines, the authors said. One is its predictability: like soulmates, A always binds to T, and C with G. This chemical linking in turn forms the famous double helix structure. By giving the letters little chemical additions, or swapping them out altogether with unnatural synthetic letters, scientists have been able to form entirely new DNA assemblies, folded into various 3D structures.

Rather than an unbreakable, immutable chain, DNA components are more like Japanese origami paper, or Lego blocks. While they cant make every single shapetry building a completely spherical Death Star out of Legothe chemistry is flexible enough that scientists can tweak its structure, stiffness, and coiling by shifting around the letters or replacing them with entirely new ones.

In the late fall of 1980, Dr. Nadrian Seeman was relaxing at the campus pub at New York University when he noticed a mind-bending woodcut, Depth, by MC Escher. With a spark of insight, he realized that he could form similar lattice shapes using DNA, which would make it a lot easier for him to study the molecules shape. More than a decade later, his lab engineered the first artificial 3D nanostructurea cube made out of DNA molecules. The field of DNA nanotechnology was born.

Originally considered a novelty, technologists rushed to make increasingly complex shapes, such as smiley faces, snowflakes, a tiny world map, and more recently, the worlds smallest playable tic-tac-toe set. It wasnt just fun. Along the way, scientists uncovered sophisticated principles and engineering techniques to shape DNA strands into their desired structures, forming a blueprint of DNA engineering.

Then came the DNA revolution. Reading and writing the molecule from scratch became increasingly cheaper, making it easier to experiment with brand-new designs. Additional chemical or fluorescent tags or other modifications gave scientists a direct view of their creations. Rather than a fringe academic pursuit, DNA origami became accessible to most labs, and the number of devices rapidly explodeddevices that can sense, transmit, and generate mechanical forces inside cells.

If you put together these three main components of mechanical devices, you begin to get hammers and cogs and wheels and you can start building nano machines, said Salaita.

Salaita is among several dozen labs demoing the practical uses of DNA devices.

For example, our cells are full of long-haul driver proteins that carry nutrients and other cargo throughout their interior by following specific highways (it eerily looks like a person walking down a tightrope). Just as too much traffic damages our roadways, changes in our cells logistical players can also harm the cells skeleton. Here, scientists have used DNA handles to measure force-induced changes like stretching, unfolding, and rupture of molecules involved in our cells distribution system to look for signs of trouble.

Then there are DNA tension sensors, which act like scales and other force gauges in our macroscopic world. Made up of a stretchable DNA spring to extend with force, and a fluorescent ruler that measures the extension, each sensor is anchored at one end (generally, the glass bottom of a Petri dish) and binds to a cell at the other. If the pulling force exceeds a certain threshold, the spring unfolds and quenches the fluorescent light in the ruler, giving scientists a warning that the cellular tugging is too strong.

The work may sound abstruse, but its implications are plenty. One is for CAR-T, the revolutionary cancer treatment that uses gene therapy to amp up immune cells with better graspers to target tumor cells. The kiss of death between graspers and tumors are extremely difficult to measure because its light and fleeting. Using a DNA tension sensor, the team was able to track the force during the interaction, which could help scientists engineer better CAR-T therapies. A similar construct, the DNA tension gauge tether, irreversibly ruptures under too much force. The gauge is used to track how stem cells develop into brain cells under mechanical forces, and how immune cells track down and recognize foreign invasion.

[Immune] T cells are constantly sampling cells throughout your body using these mechanical tugs. They bind and pull on proteins on a cells surface and, if the bond is strong, thats a signal that the T cell has found a foreign agent, explained Salaita. DNA devices provide an unprecedented look at these forces in the immune system, which in turn could predict how strongly the body will mount an immune response.

To the authors, however, the most promising emerging DNA devices dont just observethey can also generate forces. DNA walkers, for example, uses DNA feet to transport (and sort) molecular cargo while walking down a track also made of DNA strands. When the feet bind to the track (A to T, C to G), it releases energy that propel the walker forward.

Even more exciting are self-assembling DNA machines. The field has DNA-based devices that transmit, sense and generate mechanical forces, the authors said. But eventually, their integration will produce nanomachines that exert mechanical control over living systems.

As costs keep dropping, the authors believe well witness even more creative and sophisticated DNA nanomachines.

Several hiccups do stand in the way. Like other biomolecules, foreign DNA can be chopped up by the bodys immune system as an invader. However, the team believes that the limitation wont be a problem in the next few years as biochemistry develops chemically-modified artificial DNA letters that resist the bodys scissors.

Another problem is that the DNA devices can generate very little forceless than a billionth the weight of a paperclip, which is a little too low to efficiently control forces in our cells. The authors have a solution here too: coupling many force-generating DNA units together, or engineer translators that can turn electrical energy into mechanical forcesimilar to the way our muscles work.

Fundamentally, any advancements in DNA mechanotechnology wont just benefit medicine; they will also feed back into the design of nanomaterials. The techniques, tools and design principlesare not specific to DNA, the authors said. Add in computer-aided design templates, similar to those used in 3D printing, and potentially anyone can dream up a nano-machine design and make it a reality, said Salaita.

Image Credit: Emory University. DNA mechanotechnology expands the opportunities for research involving biomedicine and materials sciences, says Khalid Salaita, right, professor of chemistry at Emory University and co-author of the article, along with Aaron Blanchard, left, a graduate student in the Salaita Lab.

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DNA Nanomachines Are Opening Medicine to the World of Physics - Singularity Hub

PTPRU, As A Tumor Suppressor, Inhibits Cancer Stemness By Attenuating | OTT – Dove Medical Press

Jiayi Gu,1,* Zhiqi Zhang,2,* Tingyuan Lang,3,* Xinlin Ma,1 Linxi Yang,1 Jia Xu,1 Cong Tian,4 Kun Han,4 Jiangfeng Qiu1

1Department of Gastrointestinal Surgery, Renji Hospital Shanghai Jiao Tong University School of Medicine, Shanghai 200127, Peoples Republic of China; 2Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth Peoples Hospital, Shanghai 200233, Peoples Republic of China; 3Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital and Chongqing Cancer Institute and Chongqing Cancer Hospital, Chongqing 400030, Peoples Republic of China; 4Department of Medical Oncology, Shanghai Jiao Tong University Affiliated Sixth Peoples Hospital, Shanghai 200233, Peoples Republic of China

*These authors contributed equally to this work

Correspondence: Jiangfeng QiuDepartment of Gastrointestinal Surgery, Renji Hospital Shanghai Jiao Tong University School of Medicine, No.160 Pujian Road, Pudong New Area, Shanghai 200127, Peoples Republic of ChinaTel +86 138 1687 3899Fax +86 21 6373 0455Email qif0228@126.com

Background: PTPRU is an important signaling molecule that regulates a variety of cellular processes; however, the role of PTPRU in cancer development has remained elusive. Here, we report that PTPRU serves as a tumor suppressor that inhibits cancer stemness by attenuating Hippo/YAP signaling pathway.Methods: Primary cancer cells and cell line cells were used in the study. The gene expression data were downloaded from R2 analysis and visualization platform and KaplanMeier analysis was performed to study the relationship between survival and PTPRU expression. qRT-PCR and Western blot were employed to study the expression of target genes in tissues and cells. Sphere and colony formation, proliferation, migration activities and the expression of stem cell and EMT markers were employed for characterizing the stemness. Gene manipulation was achieved by lentivirus-mediated gene delivery system. Luciferase reporter gene assay was used to study the transcriptional activity of the promoter, and ChIP-qPCR was employed to study the target binding sequence of the protein. Spearman correlation analysis was performed to study the correlation between two genes. Students t-test was used for determination of the significance between two experimental groups.Results: PTPRU is downregulated in colorectal and gastric cancer tissues and cancer stem cells. High expression of PTPRU predicts poor prognosis. Overexpression of PTPRU attenuates the stemness of gastric cancer stem cells and knockdown of PTRPU improves the maintenance of the stemness of cancer stem cells. Mechanistic analysis showed that PTPRU inhibits Hippo/YAP signaling by suppressing the expression of YAP in a transcriptional level. Overexpression of YAP restored PTPRU-induced inhibited stemness of gastric cancer stem cells.Conclusion: PTPRU serves as a tumor suppressor that inhibits the stemness of cancer stem cell by inhibiting Hippo/YAP signaling pathway.

Keywords: PTPRU, cancer stem cells, Hippo/YAP signaling, gastric cancer, colorectal cancer

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License.By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

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PTPRU, As A Tumor Suppressor, Inhibits Cancer Stemness By Attenuating | OTT - Dove Medical Press

Keeping the Gene Expression of Sex Chromosomes in Balance – Technology Networks

Researchers at Karolinska Institutet in Sweden have uncovered a chromosome-wide mechanism that keeps the gene expression of sex chromosomes in balance in our cells. The findings shed light on molecular reasons for early miscarriage and could be important for the emerging field of regenerative medicine. The study is published in Nature Structural and Molecular Biology.

The genes in our cells are packaged into 23 large units of DNA called chromosomes. The sex chromosomes, X and Y, differ from all other chromosomes in that they are only present as one active copy per cell instead of two. This renders a copy-number imbalance between genes located on sex chromosomes and the rest of our genome. Now researchers at Karolinska Institutet have figured out how our cells manage to double the expression of X-chromosome genes to achieve balance.

By examining gene expression dynamics in fine detail in female and male embryonic and somatic cells, the researchers found that genes on the X chromosome produced waves of gene products at a faster tempo than other chromosomes.

"The X chromosome generates 'bursts' of gene expression at higher rate that other chromosomes, pointing to the involvement of special DNA elements called enhancers in maintaining an elevated X-chromosome expression" says Bjrn Reinius, the principal investigator at the Department of Medical Biochemistry and Biophysics, who directed the study.

In female cells, carrying two X chromosomes, the increased tempo established on one X-chromosome copy during the same developmental window in which the second X-copy became inactivated. 'X inactivation' is a previously characterized mechanism that keeps one X chromosome silent in women, resulting in patches of cells expressing either the maternal or paternal copy. Male cells, carrying only one X chromosome, instead maintained a constantly fast rhythm of expression throughout developmental phases and cell types.

"Failure to establish X-chromosome dosage compensation during the early female embryogenesis is lethal and leads to early spontaneous abortion" Reinius says. "With the new knowledge, we better understand how the cells' gene expression network becomes destabilised."

According to the researchers, the findings represent a breakthrough in understanding sex-chromosome gene regulation. Understanding these chromosome-wide mechanisms could also be important in the field of regenerative medicine, since the reprogramming of cells may disturb the X-chromosome dosage balance in a gender-specific manner.

Reference: Larsson, Coucoravas, Sandberg and Reinius. 2019.X-chromosome upregulation is driven by increased burst frequency. Nature Structural and Molecular Biology. DOI: https://doi.org/10.1038/s41594-019-0306-y.

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The Curious Incident of the Blind Dog in the Night-time – Technology Networks

Creating an effective gene therapy for inherited diseases requires three key steps. First, scientists must identify and characterize the disease. Second, they must find the gene responsible. And finally, they must find a way to correct the impairment.

Four years ago, a team from the University of Pennsylvania, in collaboration with a group from Japan, ticked the first box of that checklist with regard to a form of congenital night blindness in dogs. Now, in a paper in the journal Scientific Reports, they announce success in the second stage: theyve identified the gene responsible.

We have indeed nailed down the exact genetic mutation that is causing this disease, says Keiko Miyadera, an assistant professor of ophthalmology at Penns School of Veterinary Medicine and the senior author on the paper. The next stage is to work on treating this condition; that is to come, and were very excited about it.

People with congenital stationary night blindness (CSNB) have virtually normal vision during the day, but struggle to make out objects in dim light. The heritable condition is present from birth and can arise from mutations in a number of genes. While the modern world is generally well-lit, this form of blindness can seriously impact quality of life in areas where artificial lighting is not as readily available.

In a 2015 publication in the journal PLOS ONE, a team including Miyadera and Gustavo Aguirre, a professor of ophthalmology and medical genetics at Penn Vet, and Rueben Das, then of Penn Vet and now of Penns Perelman School of Medicine, in collaboration with a team led by Mie Universitys Mineo Kondo, announced that they had, for the first time, found a form of true CSNB in dogs.

In the current work, the researchers continued their collaboration, this time working to identify the genetic mutation responsible. Taking advantage of relatively affordable genome sequencing technology, the team performed a genome-wide association study to narrow down the candidate genetic regions potentially involved.

Using a chip capable of identifying single nucleotide changes at 170,000 points in a dogs genome, the researchers studied 12 dogs with this form of CSNB and 11 unaffected dogs. All of the animals came from a closely related family, helping the differences between them stand out.

That analysis narrowed their target to a region of the genome roughly 4 million nucleotide basepairs in sizestill too large to search gene by gene. Instead, they carried out whole genome sequencing and used the results to compare to an international dataset containing genomic information from more than 250 dogs and looked for genes in which affected dogs had two copies of a mutation, carriers had one, and other dogs had none.

We found a mutation that was quite convincing, says Miyadera. The mutation affects the LRIT3 gene, involving a deletion of one basepair, causing the resulting protein to be truncated. Notably, LRIT3 mutations have also been implicated in CSNB in people.

In its normal form, LRIT3 ensures that a molecular channel protein, TRPM1, is properly localized at the tip of a cell type adjacent to the retinas light-sensing photoreceptor cells. This secondary layer of retinal neurons, called ON bipolar cells, relay signals from the photoreceptors on their path to the brain. The mutation appears to specifically affect those ON bipolar cells that are associated with rod cellsthose that kick in strongly allowing vision in dim light.

Once they had zeroed in on the LRIT3 mutation, they were able to firm up the evidence that it was the gene responsible, examining tissue from affected dogs and examining how having a normal versus mutant LRIT3 affected the cell and protein markers and expression of TRPM1 in laboratory experiments.

While the mutation affects the function of the ON bipolar cells, the researchers found that the structure of the retina appeared to be relatively unaffected by the mutation.

Thats critical for developing a gene therapy, says Aguirre. If the structure isnt in place, youre not going to be able to restore vision with that approach.

The team is already at work designing a gene therapy approach to correcting the mutation. The effort entails a different challenge from previous forms of blindness the group has worked on, as targeting the ON bipolar cells requires approaching the retina at a different layer that is not as readily accessible as the photoreceptor cells. Whats unique about this area of work is that we are trying to target a cell type that has been under-utilized as a therapeutic target before, says Miyadera.

As a result, the researchers hope their work may give rise to strategies for treating other conditions involving the ON bipolar cell layer.

Das, R.G. et al. (2019) Genome-wide association study and whole-genome sequencing identify a deletion in LRIT3 associated with canine congenital stationary night blindness. Scientific Reports. DOI: https://doi.org/10.1038/s41598-019-50573-7

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.

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The Curious Incident of the Blind Dog in the Night-time - Technology Networks

Cancer Of The Blood May Become Deadlier Than It Was – Science Times

(Photo : madartzgraphics)

New research shows that two types of cell mutation present in the blood are making them more harmful than they already are. The research team at Cold Spring Harbor Laboratory has recently identified two types of cell mutations. These two types of cell mutations can enhance the effect of the other cells and develop a strain of a deadlieracute myeloid leukemia(AML).

Omar Abdel-Wahab of the Memorial Sloan Kettering Cancer Center collaborated with Adrian Krainer, a CSHL Professor. The two presented a detailed explanation of how the mutations of the IDH2 and SRSF2 genes built the unexpected partnership that gave birth to the worst form of the AML to date.

In their report, the mutation of the IDH2 gene enhances the effect of the SRSF2 mutation preventing the maturity of red and white blood cells. Both types of cells are what every AML patient needs to fight the disease. The team is currently working on finding a way to make this so-called "partnership" stop, hoping to find a cure to one of the most potent forms of blood cancer.

"We discovered such partnership while we were evaluating the data of patients from theCancer Genome Atlas," said. Abdel-Wahab, an oncologisthaematologist. They found out that in the cases of patients who died of AML, both mutations were present.

Knowledge of the two-cell mutation has been known before this research. However, what people knew was that both are involved in exhibiting symptoms of cancer. In most cases, however, what causes the symptoms may not necessarily be the cause ofcancer.

"A mutation in the cells of a sick patient does not necessarily show its direct connection to the disease," Krainer said.

To find out if the mutations in theSRSF2and IDH2 are indeed at work to develop AML, the team of Krainer and Abdel-Wahab worked together in Krainer's lab. Their detailed findings have recently been published in the journal, Nature.

The SRSF2 gene was identified to cause errors in RNA splicing. The splicing process converts RNA to understandable instructions for particular cells in the body. Errors in this process could lead to serious cell malfunctions. At first, the researchers did not consider that the splicing could lead to AML as the mutations were only present in 1% of AML patients. However, further research showed that mutation occurs 11% of the time in AML patients.

Further experiments in the lab revealed the severity of the splicing errors caused by SRSF2, which was further enhanced by the IDH2 mutation. This results in an even more defective set of blood cells.

"In some way, these two defective genes become cooperative of each other," Krainer said. This interdependence has resulted in a lot of deaths, but knowledge of it will only lead to points of intervention.

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Cancer Of The Blood May Become Deadlier Than It Was - Science Times

New Viral Vector Improves Sickle Cell Gene Therapy – DocWire News

A unique, improved viral vector for use in treating sickle cell disease with gene therapy has recently been created by researchers from the National Institutes of Health (NIH). When tested in animal models, this vector was able to incorporate correct genes into bone marrow stem cells up to 10 times more efficiently than current vectors. This new viral vector also has a carrying capacity up to six times higher than the conventional vector, as per the researchers. The use of this new technique could potentially make gene therapy more effective and prevalent in treating sickle cell disease, which affects roughly 100,000 people in the US and millions globally. The NIH teams findings were published on October 2 in the journal Nature Communications.

Our new vector is an important breakthrough in the field of gene therapy for sickle cell disease, said senior author John Tisdale, MD, chief of the Cellular and Molecular Therapeutic Branch at the National Heart, Lung, and Blood Institute (NHLBI). Its the new kid on the block and represents a substantial improvement in our ability to produce high capacity, high efficiency vectors for treating this devastating disorder.

In gene therapy, the viral vector serves as a delivery vehicle that leverages the viruss innate ability to infiltrate host cells and administer genetic material. The vector is modified to carry a beneficial gene that will induce therapeutic effects in the patient, often by counteracting a genetic mutation.

Patients with sickle cell disease have an inherited mutation in the beta-globin gene, resulting in a faulty hemoglobin structure that yields sickle-shaped red blood cells. This shape causes the blood cells to stick to blood vessel walls, causing pain, anemia, blockage, organ damage, and premature death. Gene therapy for sickle cell involves the modification of bone marrow hematopoietic stem cells. These blood-producing cells are altered to possess a normal copy of the beta-globin gene in a lab and are then reinfused into the patient, ultimately inducing the production of healthy red blood cells.

Though this approach has been effective, Tisdale notes that there is always room for improvement in such treatments. He compares this new viral vector to a new model of a car that is easier and more scalable to produce.

Researchers have been developing these beta-globin vectors in a reverse structural orientation for over 30 years. This approach entails that the genes incorporated into the virus are translated from right to left by the enzymes, analogous to a sentence being read backward. This is done due to the sensitive expression of intron 2, a key molecular component of the vector that is required for high beta-globin expression. This intron gets excluded during the normal vector preparation process if it is not oriented in this reverse manner.

Gene therapy studies that incorporate these reverse-oriented vectors for sickle cell disease and beta-thalassemia, a similar inherited blood disease, have been encouraging thus far. The researchers note that this complicated gene translation process has made both the preparation of the vector and the gene-transfer efficiency more challenging, however.

Roughly 10 years ago, Tisdale worked with Naoya Uchida, MD, PhD, a staff scientist in his lab, to find an improved beta-globin delivery vehicle. They found a unique way to leave the intron 2 intact by creating a new forward-oriented beta-globin vector. Unlike the previously used, reverse-oriented vector, this novel vehicle is read left to right in typical fashion. Tisdale notes that this simplifies the translation of this genetic information into a tangible protein compound, like beta-globin.

These unique vectors were used in mouse and monkey models, with the results being compared to reverse-oriented vectors. The NIH team found that their vector delivered a viral load of up to six times more therapeutic beta-globin genes than the traditional vector and had four to ten times higher transduction efficiency (ability to incorporate therapeutic genes into repopulating bone marrow cells).

In addition, these new vectors remained in place four years after transplantation, supporting the longevity of this approach. This forward-oriented vector can also be produced in greater quantities than the traditional vector, cutting time and costs that come with industrial production.

Our lab has been working on improving beta-globin vectors for almost a decadeand finally decided to try something radically differentand it worked, Tisdale concluded. These findings bring us closer to a curative gene therapy approach for hemoglobin disorders.

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AIDS Pathogens: How Do They Multiply In The Body? – Science Times

Staff ReporterOct 04, 2019 06:23 AM EDT

To improve the treatment of a disease, scientists must know its root. A European research team led by Prof. Christian Eggeling from the Friedrich Schiller University Jena, the University of Oxford, and the Leibniz Institute of Photonic Technology (Leibniz IPHT) has succeeded in using high-resolution imaging to make the spread of theHI virusbetween living cells visible by the millisecond. The team has also identified the molecules the virus needs to replicate itself. Using theSTEDsuper resolution fluorescence microscopy, the research team was able to provide proof of the lipid environment where the AIDS pathogen replicates itself.

"Thestudyprovides a method of investigation to prevent the multiplication of such HI virus in the body," Eggeling said. The results of their study were published in the Science Advances journal on October 2, 2019.

In the study, the researchers discussed how they focused on the slice through which the Human Immunodeficiency Virus (HIV) emerges after the cell has been infected. They looked into the plasma membrane of the cell from which it emerged. The protein Gag was used as a marker to coordinate the process involved in the maturation process of the virus.

"We have identified that the decisive process of replication of infected cells happen where the protein Gag accumulates," Christian Eggeling explains. Looking further into this site, the researchers were able to identify that the HI virus interacts with certain lipids. Although it has been identified before, the study provides proof that the interaction happens both in infected and living cells in the body.

"This new discovery allows us to put together an antiviral drug,"Eggelingnoted. Another crucial discovery is the identification of molecules that the HI virus needs to leave one cell to infect another. With technology, this process can be followed, allowing scientists to prevent the spread of the virus from happening.

The team is now looking at developing antibodies that attack these identified molecules to suppress the virus. "The team wants the antibodies to work its magic medically, but they also want to find out the biophysical interaction that happens to further enhance their efficiency," Eggeling added.

With the use of fluorescence microscopy as a tool, the team was able to follow the labeled molecules and tracked them in real time. It ledthe team to understanding how diseases develop in the molecular level. Eggeling is closely working with physicians and biologists to discuss how these methods can further be used for easy disease detection. More importantly, he aims to make the process more accurate to hopefully prevent the diseases from spreading.

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AIDS Pathogens: How Do They Multiply In The Body? - Science Times

Identification of more than 200K Cancer Neoantigens Could Lead to New Cancer Vaccines – Clinical OMICs News

Scientists at Arizona State Universitys Biodesign Institute now report on the identification of more than 200,000 cancer neoantigens, which could feasibly lead to the development of broad-spectrum cancer vaccines, as well as tumor type-specific treatments or patient-personalized vaccines. In a cancer cell, it turns out that all levels of information transfer from DNA to RNA to protein become more error-prone, said research lead Stephen Albert Johnston, Ph.D., center director and professor, Biodesign Center for Innovations in Medicine. We proposed that these mistakes made in cancer cells may also be the source to make a cancer vaccine.

Johnston and his team at the Biodesign Institute have spent more than a decade working towards the goal of developing a universal vaccine that can prevent cancer. They report on their latest studies in Scientific Reports, in a paper titled, RNA Transcription and Splicing Errors as a Source of Cancer Frameshift Neoantigens for Vaccines.

The success of checkpoint inhibitor therapy against cancer is largely attributed to activation of the patients immune response to tumor neoantigens that result from DNA mutations in the cancer cells, the authors explained. However, while checkpoint inhibitor immunotherapies are revolutionizing how we treat cancer, about 5080% of patients with even the most responsive tumor types wont respond well to treatment. A surprising finding in the analysis of these patients was that one of the best correlates of response has been the total number of neoantigens in the tumor, the team stated. The realization that these DNA mutations have such immunological importance has accelerated research efforts to develop personal cancer vaccines. Its a promising approach, but in reality, a major problem is that the majority of tumors will not have enough neoantigen-generating mutations to sustain development of a personalized vaccine.

With this in mind, the researchers set out to look for an alternative source of neoantigens that could possibly broaden the scope of neoantigen-based cancer vaccines. They were particularly interested in how disrupted RNA processes can lead to the production of frameshift (FS) mutated peptides, and the exposure of these peptides to the immune system. As they pointed out, In the process of becoming a tumor, not only does the DNA mutation rate increase with faster cell divisions, but also there is a disruption of basic cellular functions, including RNA transcription, splicing, and the quality control system on peptides.

They reasoned that frameshift variants produced by errors in RNA processing might be a source of cancer neoantigens, and they also assumed that there is a general increase in error rates in cancer cells. For the most part these errors can be managed and cleaned up by the cells own quality control machinery. However, as cancer progresses, these mutated peptides can build up and swamp the cells ability to deal with them, so aberrant proteins are then exposed and recognized by the immune system.

These overwhelm the quality control systems of a cell, producing mistakes in RNA and proteins that are released from the cancer cell, and the immune system can respond to, said Johnston.

To quickly identify frameshift and splicing mutations, Johnstons research team designed an array to detect all possible predicted frameshift peptides that any tumor cell could potentially produce. They custom-build this frameshift array, which ended up containing almost 400,000 frameshift peptides, and screened these against the blood samples of cancer patients (and healthy samples as a control) to look for antibodies against the peptides.

We analyzed the specific IgG reactivities to these FSPs in 64 noncancer control samples and a total of 85 cancers from five different late-stage cancer types with 17 samples each (LC: lung cancer, BC: breast cancer, GBM: glioblastoma, GC: gastric cancer, PC: pancreatic cancer) and 12 stage I pancreatic cancer samples, the authors noted.

This approach is less complex than extracting, purifying, and then sequencing tumor DNA, which is the typical starting point for the development of personal cancer vaccines. Personal cancer vaccines are complicated and expensive, said Johnston. Also, only about 40% of tumors have enough mutations in the DNA to make a vaccine from. We discovered that even cold tumors at the DNA level make lots of mistakes at the RNA level. And the mistakes we focus on are frameshift peptides which are much more immunogenic than the point mutations used in personal cancer vaccines. Most importantly, we can make off-the shelf vaccines for therapeutic or even preventative vaccines which will be much less expensive.

The results of their screens indicated that all five cancer types, with the exception of glioblastoma, had significantly more peptides reacting with antibodies in the cancer patients than controls. There were also three basic patterns seen among patients with each cancer type. First, the vast majority of the frameshift peptides (6980%) were personal, or unique to that individual. Second, about 16% to 19% of the positive peptides were shared between two samples within the same cancer type, and thirdly, 1.56.9% were shared between three or more samples (with gastric cancer having the highest, at 6.9%). Strikingly, one of the hardest to treat cancers, glioblastoma, had the greatest potential for personalized vaccines. Of the 17 glioblastoma patient samples studied, each patient had 5,800 frameshift peptides, and of these, 4,500 were unique to that patient.

The team wanted to see how the frameshift mutations compared between early- and late-stage cancers. A comparison of the 20,000 peptides that they identified in late-stage and stage 1 pancreatic cancer showed little overlap, implying that a vaccine for early-stage cancer would have to be different to that for late-stage cancer.

Interestingly, studies in mouse models found that the newly discovered antigens were protective against both breast cancer and melanoma. Johnstons group has pioneered genetic immunization using gene gun technology, which they used for their experiments to shoot gold nanoparticles containing the most promising vaccines. In a typical experiment, six-week-old mice received one genetic immunization in the pinna of the ear. After four weeks, they were challenged with cancer-causing cells, and then twice received booster shots, two days apart. The results showed that the prototype vaccines could all significantly delay or even prevent tumor growth or progression. Most importantly, in the mouse vaccine challenges, they found that pooling multiple frameshift peptides resulted in a more effective vaccine, with additive effects further delaying tumor growth.

The team has used their work to date as the basis for a large preclinical trial, in dogs, funded by the Open Philanthropy Project, which is evaluating a vaccine candidate designed to be a broadly protective, prophylactic pan-cancer vaccine. Johnston has also established a spinout company, Calviri, to continue cancer vaccine development.

From their screening results and analyses of the different cancer samples, and the mouse cancer vaccine challenges, the Johnston group now has a top 100 peptide list for each of the five human cancers. We strongly believe that the data presented, as well as more to be submitted, support bringing FS antigen cancer vaccines to clinical trial, the authors wrote. We have recently initiated a large dog clinical trial of a pan-cancer prophylactic vaccine and will soon submit protocols for both dog and human therapeutic trials of cancer-type specific vaccines.

Johnston acknowledged that even optimistically it would be five to 10 years before human use. However, he concluded, This is probably the only approach to a broadly preventative cancer vaccine, so we feel we have to try it. The implications of success would be quite largefor dogs and people.

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Identification of more than 200K Cancer Neoantigens Could Lead to New Cancer Vaccines - Clinical OMICs News