Tag Archives: cells

Could synergistic action of engineered nanoparticles have a health impact?

Synergistic action can be difficult to study especially when you’re looking at nanoparticles which could be naturally occurring and/or engineered. I believe this study is focused on engineered nanoparticles (ENPs) and I think it’s the first one I’ve seen that examines synergistic action of any kind. So, bravo to the scientists for tackling a very ambitious project.

An October 1, 2020 news item on phys.org describes this work from Denmark,

Nanoparticles are used in a wide range of products and manufacturing processes because the properties of a material can change dramatically when the material comes in nano-form.

They can be used, for example, to purify wastewater and to transport medicine around the body. They are also added to, for example, socks, pillows, mattresses, phone covers and refrigerators to supply the items with an antibacterial surface.

Much research has been done on how nanoparticles affect humans and the environment and a number of studies have shown that nanoparticles can disrupt or damage our cells.

This is confirmed by a new study that has also looked at how cells react when exposed to more than one kind of nano particle at the same time.

An October 1, 2020 University of Southern Denmark press release (also on EurekAlert) by Birgitte Svennevig, which originated the news item, provides more insight into the research,

The lead author of the study is Barbara Korzeniowska from the Department of Biochemistry and Molecular Biology at SDU. The head of research is Professor Frank Kjeldsen from the same department.

His research into metal nanoparticles is supported by a European Research Grant of DKK 14 million.

“Throughout a lifetime, we are exposed to many different kinds of nano-particles, and we should investigate how the combination of different nano-particles affects us and also whether an accumulation through life can harm us,” says Barbara Korzeniowska.

She herself became interested in the subject when her little daughter one day was going in the bathtub and got a rubber duck as a toy.

– It turned out that it had been treated with nano-silver, probably to keep it free of bacteria, but small children put their toys in their mouths, and she could thus ingest nano-silver. That is highly worrying when research shows that nano-silver can damage human cells, she says.

In her new study, she looked at nano-silver and nano-platinum. She has investigated their individual effect and whether exposure of both types of nanoparticles results in a synergy effect in two types of brain cells.

– There are almost no studies of the synergy effect of nano particles, so it is important to get started with these studies, she says.

She chose nano-silver because it is already known to be able to damage cells and nano-platinum, because nano-platinum is considered to be so-called bio-inert; i.e. has a minimal interaction with human tissue.

The nanoparticles were tested on two types of brain cells: astrocytes and endothelial cells. Astrocytes are supporter cells in the central nervous system, which i.a. helps to supply the nervous system with nutrients and repair damage to the brain. Endothelial cells sit on the inside of the blood vessels and transport substances from the bloodstream to the brain.

When the endothelial cells were exposed to nano-platinum, nothing happened. When exposed to nano-silver, their ability to divide deteriorated. When exposed to both nano-silver and nano-platinum, the effect was amplified, and they died in large numbers. Furthermore, their defense mechanisms decreased, and they had difficulty communicating with each other.

– So even though nano-platinum alone does not do harm, something drastic happens when they are combined with a different kind of nano-particle, says Frank Kjeldsen.

The astrocytes were more hardy and reacted “only” with impaired ability to divide when exposed to both types of nano-particles.

An earlier study, conducted by Frank Kjeldsen, has shown a dramatic synergy effect of silver nanoparticles and cadmium ions, which are found naturally all around us on Earth.

In that study, 72 % of the cells died (in this study it was intestinal cells) as they were exposed to both nano-silver and cadmium ions. When they were only exposed to nano-silver, 25% died. When exposed to cadmium ions only, 12% died.

We are involuntarily exposed

– Little is known about how large concentrations of nano-particles are used in industrial products. We also do not know what size particles they use – size also has an effect on whether they can enter a cell, says Barbara Korzeniowska and continues:

– But we know that a lot of people are involuntarily exposed to nano-particles, and that there can be lifelong exposure.

There are virtually no restrictions on adding nanoparticles to products. In the EU, however, manufacturers must have an approval if they want to use nanoparticles in products with antibacterial properties. In Denmark, they must also declare nano-content in such products on the label.

Here’s a link to and a citation for the paper,

The Cytotoxicity of Metal Nanoparticles Depends on Their Synergistic Interactions by Barbara Korzeniowska, Micaella P. Fonseca, Vladimir Gorshkov, Lilian Skytte, Kaare L. Rasmussen, Henrik D. Schrøder, Frank Kjeldsen. Particle Volume 37, Issue 8, August 2020,. 2000135 DOI: https://doi.org/10.1002/ppsc.202000135 First published: 06 July 2020

This paper is behind a paywall.

Membrane stretching as a new transport mechanism for nanomaterials

This work comes from Catalonia, Spain by way of a collaboration between Chinese, German, and, of course, Spanish scientists. From a December 12, 2018 Universitat Rovira i Virgili press release (also on EurekAlert),

Increasing awareness of bioeffects and toxicity of nanomaterials interacting with cells puts in focus the mechanisms by which nanomaterials can cross lipid membranes. Apart from well-discussed energy-dependent endocytosis for large objects and passive diffusion through membranes by solute molecules, there can exist other transport mechanisms based on physical principles. Based on this hypothesis, the team of theoretical physics at Universitat Rovira i Virgili in Tarragona, led by Dr. Vladimir Baulin, designed a research project to investigate the interaction between nanotube and lipid membranes. In computer simulations, the researchers studied what they call a “model bilayer”, composed only by one type of lipids. Based on their calculations, the team of Dr. Baulin observed that ultra -short nanotube (10nm length) can insert perpendicularly to the lipid bilayer core.

They observed that these nanotubes stay trapped in the cell membrane, as commonly accepted by the scientific community. But a surprise appears when they stretched their model cell membrane, then inserted nanotubes which were trapped in the bilayer, suddenly started to escape from the bilayer on both sides. This means that it is possible to control the transport of nanomaterial across a cell membrane by tuning the membrane tension.

This is where Dr. Baulin contacted Dr. Jean-Baptiste Fleury at the Saarland University (Germany) to confirm this mechanism and to study experimentally this tension-mediated transport phenomena. Dr. Fleury and his team, designed a microfluidic experiment with a well-controlled phospholipid bilayer, an experimental model for cell membranes and added ultra-small carbon nanotubes (10nm in length) in solution. The nanotubes had an adsorbed lipid monolayer that guarantees their stable dispersion and prevent their clustering. Using a combination of optical fluorescent microscopy and electrophysiological measurements, the team of Dr. Fleury could follow individual nanotube crossing a bilayer and unravel their pathway on a molecular level. And as predicted by the simulations, they observed that nanotubes inserted into the bilayer by dissolving their lipid coating into the artificial membrane. When a tension of 4mN/m was applied to the bilayer, nanotubes spontaneously escaped the bilayer just in few milliseconds, while at lower tensions nanotubes remain trapped inside the membrane.

This discovery of translocation of tiny nanotubes through barriers protecting cells, i.e. lipid bilayer, may raise concerns about safety of nanomaterials for public health and suggest new mechanical mechanisms to control the drug delivery.

Caption: Nanotubes trapped inside the membrane. Credit: © URV

Here’s a link to and a citation for the paper,

Tension-Induced Translocation of an Ultrashort Carbon Nanotube through a Phospholipid Bilayer by Yachong Guo, Marco Werner, Ralf Seemann, Vladimir A. Baulin, and Jean-Baptiste Fleury. ACS Nano, Article ASAP DOI: 10.1021/acsnano.8b04657 Publication Date (Web): November 19, 2018

Copyright © 2018 American Chemical Society

This paper is behind a paywall.

Cellulose biosensor heralds new bioimaging approach to tissue engineering

I keep an eye on how nanocellulose is being used in various applications and I’m not sure that this cellulose biosensor quite fits the bill as nanocellulose, nonetheless, it’s interesting and that’s enough for me. From a December 12, 2018 Sechenov University (Russia) press release on EurekAlert,

I.M. Sechenov First Moscow State Medical University teamed up together with Irish colleagues to develop a new imaging approach for tissue engineering. The team produced so-called ‘hybrid biosensor’ scaffold materials, which are based on cellulose matrices labeled with pH- and calcium-sensitive fluorescent proteins. These materials enable visualization of the metabolism and other important biomarkers in the engineered artificial tissues by microscopy. The results of the work were published in the Acta Biomaterialia journal.
The success of tissue engineering is based on the use of scaffold matrices – materials that support the viability and direct the growth of cells, tissues, and organoids. Scaffolds are important for basic and applied biomedical research, tissue engineering and regenerative medicine, and are promising for development of new therapeutics. However, the ability ‘to see’ what happens within the scaffolds during the tissue growth poses a significant research challenge

“We developed a new approach allowing visualization of scaffold-grown tissue and cells by using labeling with biosensor fluorescent proteins. Due to the high specificity of labeling and the use of fluorescence microscopy FLIM, we can quantify changes in pH and calcium in the vicinity of cells,” says Dr. Ruslan Dmitriev, Group Leader at the University College Cork and the Institute for Regenerative Medicine (I.M. Sechenov First Moscow State Medical University).
To achieve the specific labeling of cellulose matrices, researchers used well-known cellulose-binding proteins. The use of extracellular pH- and calcium-sensitive biosensors allow for analysis of cell metabolism: indeed, the extracellular acidification is directly associated with the balance of cell energy production pathways and the glycolytic flux (release of lactate). It is also a frequent hallmark of cancer and transformed cell types. On the other hand, calcium plays a key role in the extra- and intracellular signaling affecting cell growth and differentiation.

The approach was tested on different types of cellulose matrices (bacterial and produced from decellularised plant tissues) using 3D culture of human colon cancer cells and stem-cell derived mouse small intestinal organoids. The scaffolds informed on changes in the extracellular acidification and were used together with the analysis of real-time oxygenation of intestinal organoids. The resulting data can be presented in the form of colour maps, corresponding to the areas of cell growth within different microenvironments.

“Our results open new prospects in the imaging of tissue-engineered constructs for regenerative medicine. They enable deeper understanding of tissue metabolism in 3D and are also highly promising for commercialisation,” concludes Dr. Dmitriev.

The researchers have provided an image to illustrate their work,

Caption: A 3D reconstruction of a cellulose matrix stained with a pH-sensitive biosensor. Credit: Dr. R. Dmitriev

Here’s a link to and a citation for the paper,

Cellulose-based scaffolds for fluorescence lifetime imaging-assisted tissue engineering by Neil O’Donnell, Irina A. Okkelman, Peter Timashev, Tatyana I.Gromovykh, Dmitri B. Papkovsky, Ruslan I.Dmitriev. Acta Biomaterialia Volume 80, 15 October 2018, Pages 85-96 DOI: https://doi.org/10.1016/j.actbio.2018.09.034


This paper is behind a paywall.

The CRISPR ((clustered regularly interspaced short palindromic repeats)-CAS9 gene-editing technique may cause new genetic damage kerfuffle

Setting the stage

Not unexpectedly, CRISPR-Cas9  or clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 can be dangerous as these scientists note in a July 16, 2018 news item on phys.org,

Scientists at the Wellcome Sanger Institute have discovered that CRISPR/Cas9 gene editing can cause greater genetic damage in cells than was previously thought. These results create safety implications for gene therapies using CRISPR/Cas9 in the future as the unexpected damage could lead to dangerous changes in some cells.

Reported today (16 July 2018) in the journal Nature Biotechnology, the study also revealed that standard tests for detecting DNA changes miss finding this genetic damage, and that caution and specific testing will be required for any potential gene therapies.

This CRISPR-Cas9 image reminds me of popcorn,

CRISPR-associated protein Cas9 (white) from Staphylococcus aureus based on Protein Database ID 5AXW. Credit: Thomas Splettstoesser (Wikipedia, CC BY-SA 4.0)[ downloaded from https://phys.org/news/2018-07-genome-crisprcas9-gene-higher-thought.html#jCp]

A July 16, 2018 Wellcome Sanger Institute press release (also on EurekAlert), which originated the news item, offers a little more explanation,

CRISPR/Cas9 is one of the newest genome editing tools. It can alter sections of DNA in cells by cutting at specific points and introducing changes at that location. Already extensively used in scientific research, CRISPR/Cas9 has also been seen as a promising way to create potential genome editing treatments for diseases such as HIV, cancer or sickle cell disease. Such therapeutics could inactivate a disease-causing gene, or correct a genetic mutation. However, any potential treatments would have to prove that they were safe.

Previous research had not shown many unforeseen mutations from CRISPR/Cas9 in the DNA at the genome editing target site. To investigate this further the researchers carried out a full systematic study in both mouse and human cells and discovered that CRISPR/Cas9 frequently caused extensive mutations, but at a greater distance from the target site.

The researchers found many of the cells had large genetic rearrangements such as DNA deletions and insertions. These could lead to important genes being switched on or off, which could have major implications for CRISPR/Cas9 use in therapies. In addition, some of these changes were too far away from the target site to be seen with standard genotyping methods.

Prof Allan Bradley, corresponding author on the study from the Wellcome Sanger Institute, said: “This is the first systematic assessment of unexpected events resulting from CRISPR/Cas9 editing in therapeutically relevant cells, and we found that changes in the DNA have been seriously underestimated before now. It is important that anyone thinking of using this technology for gene therapy proceeds with caution, and looks very carefully to check for possible harmful effects.”

Michael Kosicki, the first author from the Wellcome Sanger Institute, said: “My initial experiment used CRISPR/Cas9 as a tool to study gene activity, however it became clear that something unexpected was happening. Once we realised the extent of the genetic rearrangements we studied it systematically, looking at different genes and different therapeutically relevant cell lines, and showed that the CRISPR/Cas9 effects held true.”

The work has implications for how CRISPR/Cas9 is used therapeutically and is likely to re-spark researchers’ interest in finding alternatives to the standard CRISPR/Cas9 method for gene editing.

Prof Maria Jasin, an independent researcher from Memorial Slone Kettering Cancer Centre, New York, who was not involved in the study said: “This study is the first to assess the repertoire of genomic damage arising at a CRISPR/Cas9 cleavage site. While it is not known if genomic sites in other cell lines will be affected in the same way, this study shows that further research and specific testing is needed before CRISPR/Cas9 is used clinically.”

For anyone who’d like to better understand the terms gene editing and CRISPR-Cas9, the Wellcome Sanger Institute provides these explanatory webpages, What is genome editing? and What is CRISPR-Cas9?

For the more advanced, here’s a link and a citation for the paper,

Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements by Michael Kosicki, Kärt Tomberg, & Allan Bradley. Nature Biotechnology DOI: https://doi.org/10.1038/nbt.4192 Published 16 July 2018

This paper appears to be open access.

The kerfuffle

It seems this news has affected the CRISPR market. From a July 16, 2018 article by Cale Guthrie Weissman for Fast Company,

… CRISPR could unknowingly delete or alter non-targeted genes, which could lead to myriad unintended consequences. This is especially frightening, since the technology is going to be used in human clinical trials.

Meanwhile, other scientists working with CRISPR are trying to downplay the findings, telling STAT [a life sciences and business journalism website] that there have been no reported adverse effects similar to what the study describes. The news, however, has brought about a market reaction–at least three publicly traded companies that focus on CRISPR-based therapies are in stock nosedive. Crispr Therapeutics is down by over 6%; Editas fell by over 3%; and Intellia Therapeutics dropped by over 5%. [emphasis mine]

Damage control

Gaetan Burgio (geneticist, Australian National University)  in a July 16, 2018 essay on phys.org (originating from The Conversation) suggests some calm (Note: Links have been removed),

But a new study has called into question the precision of the technique [CRISPR gene editing technology].

The hope for gene editing is that it will be able to cure and correct diseases. To date, many successes have been reported, including curing deafness in mice, and in altering cells to cure cancer.

Some 17 clinical trials in human patients are registered [emphasis mine] testing gene editing on leukaemias, brain cancers and sickle cell anaemia (where red blood cells are misshaped, causing them to die). Before implementing CRISPR technology in clinics to treat cancer or congenital disorders, we must address whether the technique is safe and accurate.

There are a few options for getting around this problem. One option is to isolate the cells we wish to edit from the body and reinject only the ones we know have been correctly edited.

For example, lymphocytes (white blood cells) that are crucial to killing cancer cells could be taken out of the body, then modified using CRISPR to heighten their cancer-killing properties. The DNA of these cells could be sequenced in detail, and only the cells accurately and specifically gene-modified would be selected and delivered back into the body to kill the cancer cells.

While this strategy is valid for cells we can isolate from the body, some cells, such as neurons and muscles, cannot be removed from the body. These types of cells might not be suitable for gene editing using Cas9 scissors.

Fortunately, researchers have discovered other forms of CRISPR systems that don’t require the DNA to be cut. Some CRISPR systems only cut the RNA, not the DNA (DNA contains genetic instructions, RNA convey the instructions on how to synthesise proteins).

As RNA [ribonucleic acid] remains in our cells only for a specific period of time before being degraded, this would allow us to control the timing and duration of the CRISPR system delivery and reverse it (so the scissors are only functional for a short period of time).

This was found to be successful for dementia in mice. Similarly, some CRISPR systems simply change the letters of the DNA, rather than cutting them. This was successful for specific mutations causing diseases such as hereditary deafness in mice.

I agree with Burgio’s conclusion (not included here) that we have a lot more to learn and I can’t help wondering why there are 17 registered human clinical trials at this point.

Nanoparticle-based delivery platform for CRISPR-Cas9 (gene-editing technology)

A February 18, 2018 King Abdullah University of Science and Technology (KAUST; Saudi Arabia) news release (also on EurekAlert but published on Feb. 20, 2018) describes a new technology for delivering CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 into cells,

A new delivery system for introducing gene-editing technology into cells could help safely and efficiently correct disease-causing mutations in patients.

The system, developed by KAUST scientists, is the first to use sponge-like ensembles of metal ions and organic molecules to coat the molecular components of the precision DNA-editing technology known as CRISPR/Cas9, allowing efficient release of the genome-editing machinery inside the cell.

“This method presents an easy and economically feasible route to improve on the delivery problems that accompany RNA-based therapeutic approaches,” says Niveen Khashab, the associate professor of chemical sciences at KAUST who led the study. “This may permit such formulations to be eventually used for treating genetic diseases effectively in the future.”

CRISPR/Cas9 has a double delivery problem: For the gene-editing technology to work like a molecular Swiss Army knife, both a large protein (the Cas9 cutting enzyme) and a highly charged RNA component (the guide RNA used for DNA targeting) must each get from the outside of the cell into the cytoplasm and finally into the nucleus, all without getting trapped in the tiny intracellular bubbles that are known as endosomes.

To solve this problem, Khashab and her lab turned to a nano-sized type of porous material known as a zeolitic imidazolate framework, which forms a cage-like structure into which other molecules can be placed. The researchers encapsulated the Cas9 protein and guide RNA in this material and then introduced the resulting nanoparticles into hamster cells.

The encapsulated CRISPR-Cas9 constructs were not toxic to the cells. And because particles in the coating material become positively charged when absorbed into endosomes, they caused these membrane-bound bubbles to burst, freeing the CRISPR-Cas9 machinery to travel to the nucleus, home to the cell’s genome. There the gene-editing technology could get to work.

Using a guide RNA designed to target a gene that caused the cells to glow green under fluorescent light, Khashab and her team showed that they could reduce the expression of this gene by 37 percent over four days with their technology. “These cage-like structures are biocompatible and can be triggered on demand, making them smart options to overcome delivery problems of genetic materials and proteins,” says the study’s first author Shahad Alsaiari, a Ph.D. student in Khashab’s lab.

The researchers’ plan to test their system in human cells and in mice, and eventually, they hope, in clinical trials.

The zeolitic imidazolate framework forms a cage-like scaffold over the CRISPR/Cas9 machinery.. Reprinted (adapted) with permission from Alsaiari, S.K., Patil, S., Alyami, M., Alamoudi, K.O., Aleisa, F.A., Merzaban, J., Li M. & Khashab, N.M. Endosomal escape and delivery of CRISPR/Cas9 genome editing machinery enabled by nanoscale zeolitic imidazolate framework. Journal of the American Chemical Society 140, 143–146 (2018). © 2018 American Chemical Society; KAUST Xavier Pita and Heno Huang ][downloaded from https://discovery.kaust.edu.sa/en/article/475/a%250adelivery-platform-for-gene-editing-technology]

Here’s a link to and a citation for the paper,

Endosomal Escape and Delivery of CRISPR/Cas9 Genome Editing Machinery Enabled by Nanoscale Zeolitic Imidazolate Framework by Shahad K. Alsaiari, Sachin Patil, Mram Alyami, Kholod O. Alamoudi, Fajr A. Aleisa, Jasmeen S. Merzaban, Mo Li, and Niveen M. Khashab. J. Am. Chem. Soc., 2018, 140 (1), pp 143–146 DOI: 10.1021/jacs.7b11754 Publication Date (Web): December 22, 2017

Copyright © 2017 American Chemical Society

This paper is behind a paywall.

CRISPR-Cas12a as a new diagnostic tool

Similar to Cas9, Cas12a is has an added feature as noted in this February 15, 2018 news item on ScienceDaily,

Utilizing an unsuspected activity of the CRISPR-Cas12a protein, researchers created a simple diagnostic system called DETECTR to analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance and also diagnose bacterial and viral infections. The scientists discovered that when Cas12a binds its double-stranded DNA target, it indiscriminately chews up all single-stranded DNA. They then created reporter molecules attached to single-stranded DNA to signal when Cas12a finds its target.

A February 15, 2018 University of California at Berkeley (UC Berkeley) news release by Robert Sanders and which originated the news item, provides more detail and history,

CRISPR-Cas12a, one of the DNA-cutting proteins revolutionizing biology today, has an unexpected side effect that makes it an ideal enzyme for simple, rapid and accurate disease diagnostics.

blood in test tube

(iStock)

Cas12a, discovered in 2015 and originally called Cpf1, is like the well-known Cas9 protein that UC Berkeley’s Jennifer Doudna and colleague Emmanuelle Charpentier turned into a powerful gene-editing tool in 2012.

CRISPR-Cas9 has supercharged biological research in a mere six years, speeding up exploration of the causes of disease and sparking many potential new therapies. Cas12a was a major addition to the gene-cutting toolbox, able to cut double-stranded DNA at places that Cas9 can’t, and, because it leaves ragged edges, perhaps easier to use when inserting a new gene at the DNA cut.

But co-first authors Janice Chen, Enbo Ma and Lucas Harrington in Doudna’s lab discovered that when Cas12a binds and cuts a targeted double-stranded DNA sequence, it unexpectedly unleashes indiscriminate cutting of all single-stranded DNA in a test tube.

Most of the DNA in a cell is in the form of a double-stranded helix, so this is not necessarily a problem for gene-editing applications. But it does allow researchers to use a single-stranded “reporter” molecule with the CRISPR-Cas12a protein, which produces an unambiguous fluorescent signal when Cas12a has found its target.

“We continue to be fascinated by the functions of bacterial CRISPR systems and how mechanistic understanding leads to opportunities for new technologies,” said Doudna, a professor of molecular and cell biology and of chemistry and a Howard Hughes Medical Institute investigator.

DETECTR diagnostics

The new DETECTR system based on CRISPR-Cas12a can analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance as well as diagnose bacterial and viral infections. Target DNA is amplified by RPA to make it easier for Cas12a to find it and bind, unleashing indiscriminate cutting of single-stranded DNA, including DNA attached to a fluorescent marker (gold star) that tells researchers that Cas12a has found its target.

The UC Berkeley researchers, along with their colleagues at UC San Francisco, will publish their findings Feb. 15 [2018] via the journal Science’s fast-track service, First Release.

The researchers developed a diagnostic system they dubbed the DNA Endonuclease Targeted CRISPR Trans Reporter, or DETECTR, for quick and easy point-of-care detection of even small amounts of DNA in clinical samples. It involves adding all reagents in a single reaction: CRISPR-Cas12a and its RNA targeting sequence (guide RNA), fluorescent reporter molecule and an isothermal amplification system called recombinase polymerase amplification (RPA), which is similar to polymerase chain reaction (PCR). When warmed to body temperature, RPA rapidly multiplies the number of copies of the target DNA, boosting the chances Cas12a will find one of them, bind and unleash single-strand DNA cutting, resulting in a fluorescent readout.

The UC Berkeley researchers tested this strategy using patient samples containing human papilloma virus (HPV), in collaboration with Joel Palefsky’s lab at UC San Francisco. Using DETECTR, they were able to demonstrate accurate detection of the “high-risk” HPV types 16 and 18 in samples infected with many different HPV types.

“This protein works as a robust tool to detect DNA from a variety of sources,” Chen said. “We want to push the limits of the technology, which is potentially applicable in any point-of-care diagnostic situation where there is a DNA component, including cancer and infectious disease.”

The indiscriminate cutting of all single-stranded DNA, which the researchers discovered holds true for all related Cas12 molecules, but not Cas9, may have unwanted effects in genome editing applications, but more research is needed on this topic, Chen said. During the transcription of genes, for example, the cell briefly creates single strands of DNA that could accidentally be cut by Cas12a.

The activity of the Cas12 proteins is similar to that of another family of CRISPR enzymes, Cas13a, which chew up RNA after binding to a target RNA sequence. Various teams, including Doudna’s, are developing diagnostic tests using Cas13a that could, for example, detect the RNA genome of HIV.

infographic about DETECTR system

(Infographic by the Howard Hughes Medical Institute)

These new tools have been repurposed from their original role in microbes where they serve as adaptive immune systems to fend off viral infections. In these bacteria, Cas proteins store records of past infections and use these “memories” to identify harmful DNA during infections. Cas12a, the protein used in this study, then cuts the invading DNA, saving the bacteria from being taken over by the virus.

The chance discovery of Cas12a’s unusual behavior highlights the importance of basic research, Chen said, since it came from a basic curiosity about the mechanism Cas12a uses to cleave double-stranded DNA.

“It’s cool that, by going after the question of the cleavage mechanism of this protein, we uncovered what we think is a very powerful technology useful in an array of applications,” Chen said.

Here’s a link to and a citation for the paper,

CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity by Janice S. Chen, Enbo Ma, Lucas B. Harrington, Maria Da Costa, Xinran Tian, Joel M. Palefsky, Jennifer A. Doudna. Science 15 Feb 2018: eaar6245 DOI: 10.1126/science.aar6245

This paper is behind a paywall.

Stretchy optical materials for implants that could pulse light

An Oct. 17, 2016 Massachusetts Institute of Technology (MIT) news release (also on EurekAlert) by Emily Chu describes research that could lead to long-lasting implants offering preventive health strategies,

Researchers from MIT and Harvard Medical School have developed a biocompatible and highly stretchable optical fiber made from hydrogel — an elastic, rubbery material composed mostly of water. The fiber, which is as bendable as a rope of licorice, may one day be implanted in the body to deliver therapeutic pulses of light or light up at the first sign of disease. [emphasis mine]

The researchers say the fiber may serve as a long-lasting implant that would bend and twist with the body without breaking down. The team has published its results online in the journal Advanced Materials.

Using light to activate cells, and particularly neurons in the brain, is a highly active field known as optogenetics, in which researchers deliver short pulses of light to targeted tissues using needle-like fibers, through which they shine light from an LED source.

“But the brain is like a bowl of Jell-O, whereas these fibers are like glass — very rigid, which can possibly damage brain tissues,” says Xuanhe Zhao, the Robert N. Noyce Career Development Associate Professor in MIT’s Department of Mechanical Engineering. “If these fibers could match the flexibility and softness of the brain, they could provide long-term more effective stimulation and therapy.”

Getting to the core of it

Zhao’s group at MIT, including graduate students Xinyue Liu and Hyunwoo Yuk, specializes in tuning the mechanical properties of hydrogels. The researchers have devised multiple recipes for making tough yet pliable hydrogels out of various biopolymers. The team has also come up with ways to bond hydrogels with various surfaces such as metallic sensors and LEDs, to create stretchable electronics.

The researchers only thought to explore hydrogel’s use in optical fibers after conversations with the bio-optics group at Harvard Medical School, led by Associate Professor Seok-Hyun (Andy) Yun. Yun’s group had previously fabricated an optical fiber from hydrogel material that successfully transmitted light through the fiber. However, the material broke apart when bent or slightly stretched. Zhao’s hydrogels, in contrast, could stretch and bend like taffy. The two groups joined efforts and looked for ways to incorporate Zhao’s hydrogel into Yun’s optical fiber design.

Yun’s design consists of a core material encased in an outer cladding. To transmit the maximum amount of light through the core of the fiber, the core and the cladding should be made of materials with very different refractive indices, or degrees to which they can bend light.

“If these two things are too similar, whatever light source flows through the fiber will just fade away,” Yuk explains. “In optical fibers, people want to have a much higher refractive index in the core, versus cladding, so that when light goes through the core, it bounces off the interface of the cladding and stays within the core.”

Happily, they found that Zhao’s hydrogel material was highly transparent and possessed a refractive index that was ideal as a core material. But when they tried to coat the hydrogel with a cladding polymer solution, the two materials tended to peel apart when the fiber was stretched or bent.

To bond the two materials together, the researchers added conjugation chemicals to the cladding solution, which, when coated over the hydrogel core, generated chemical links between the outer surfaces of both materials.

“It clicks together the carboxyl groups in the cladding, and the amine groups in the core material, like molecular-level glue,” Yuk says.

Sensing strain

The researchers tested the optical fibers’ ability to propagate light by shining a laser through fibers of various lengths. Each fiber transmitted light without significant attenuation, or fading. They also found that fibers could be stretched over seven times their original length without breaking.

Now that they had developed a highly flexible and robust optical fiber, made from a hydrogel material that was also biocompatible, the researchers began to play with the fiber’s optical properties, to see if they could design a fiber that could sense when and where it was being stretched.

They first loaded a fiber with red, green, and blue organic dyes, placed at specific spots along the fiber’s length. Next, they shone a laser through the fiber and stretched, for instance, the red region. They measured the spectrum of light that made it all the way through the fiber, and noted the intensity of the red light. They reasoned that this intensity relates directly to the amount of light absorbed by the red dye, as a result of that region being stretched.

In other words, by measuring the amount of light at the far end of the fiber, the researchers can quantitatively determine where and by how much a fiber was stretched.

“When you stretch a certain portion of the fiber, the dimensions of that part of the fiber changes, along with the amount of light that region absorbs and scatters, so in this way, the fiber can serve as a sensor of strain,” Liu explains.

“This is like a multistrain sensor through a single fiber,” Yuk adds. “So it can be an implantable or wearable strain gauge.”

The researchers imagine that such stretchable, strain-sensing optical fibers could be implanted or fitted along the length of a patient’s arm or leg, to monitor for signs of improving mobility.

Zhao envisions the fibers may also serve as sensors, lighting up in response to signs of disease.

“We may be able to use optical fibers for long-term diagnostics, to optically monitor tumors or inflammation,” he says. “The applications can be impactful.”

Here’s a link to and a citation for the paper,

Highly Stretchable, Strain Sensing Hydrogel Optical Fibers by Jingjing Guo, Xinyue Liu, Nan Jiang, Ali K. Yetisen, Hyunwoo Yuk, Changxi Yang, Ali Khademhosseini, Xuanhe Zhao, and Seok-Hyun Yun. Advanced Materials DOI: 10.1002/adma.201603160 Version of Record online: 7 OCT 2016

© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

This paper is behind a paywall.

Watching artificial nanofibres self-sort in real-time

A May 31, 2016 news item on phys.org describes research on self-assembling fibres at Kyoto University (Japan) by referencing the ancient Greek mythological figure, Psyche,

The Greek goddess Psyche borrowed help from ants to sort a room full of different grains. Cells, on the other hand, do something similar without Olympian assistance, as they organize molecules into robust, functional fibers. Now scientists are able to see self-sorting phenomena happen in real time with artificial molecules.

The achievement, reported in Nature Chemistry, elucidates how two different types of nanofibers sort themselves into organized structures under artificial conditions.

“Basic cellular structures, such as actin filaments, come into being through the autonomous self-sorting of individual molecules, even though a tremendous variety of proteins and small molecules are present inside the cell,” says lead author Hajime Shigemitsu, a researcher in Itaru Hamachi’s lab at Kyoto University.

A May 30, 2016 Kyoto University news release (also on EurekAlert), which originated the news item, expands on the theme,

“Imagine a box filled with an assortment of building blocks — it’s as if the same type of blocks started sorting themselves into neat bundles all on their own. In living cells, such phenomena always happen, enabling accurate self-assembling of proteins, which is essential for cell functions.”

“If we are able to control self-sorting with artificial molecules, we can work toward developing intelligent, next-generation biomimics that possess the flexibility and diversity of functions that exist in a living cell.”

Study co-author Ryou Kubota explains that previous studies have already made artificial molecules build themselves into fibers — but only when there was one type of molecule around. Having a jumble of types, on the other hand, made the molecules confused.

“The difficulty in inducing self-assembly with artificial molecules is that they don’t recognize the same type of molecule, unlike molecules in the natural world. Different types of artificial molecules interact with each other and make an unsorted cluster.”

From a database of structural analyses, Hamachi and colleagues discovered a combination of nanofibers — namely a peptide-based and lipid-based hydrogelator — that would make sorted fibers without mixing with the other. They then tethered the fibers with fluorescent probes; with a type of microscope typically used in cell imaging, the team was able to observe directly and in real-time how the artificial molecules sorted themselves.

“Ultimately, this finding could help develop new materials that respond dynamically to different environments and stimuli,” elaborates Hamachi. “This insight is not only useful for materials science, but may also provide useful clues for understanding self-organization in cells.”

Here’s a link to and a citation for the paper,

In situ real-time imaging of self-sorted supramolecular nanofibres by Shoji Onogi, Hajime Shigemitsu, Tatsuyuki Yoshii, Tatsuya Tanida, Masato Ikeda, Ryou Kubota, & Itaru Hamachi. Nature Chemistry (2016) doi:10.1038/nchem.2526 Published online 30 May 2016

This paper is behind a paywall bu the researchers have made a video of the self-sorting proteins freely available,

Cutting into a cell with a nanoblade

A May 11, 2016 news item on Nanotechnology Now features a type of surgery that could aid in cell engineering,

To study certain aspects of cells, researchers need the ability to take the innards out, manipulate them, and put them back. Options for this kind of work are limited, but researchers reporting May 10 [2016] in Cell Metabolism describe a “nanoblade” that can slice through a cell’s membrane to insert mitochondria. The researchers have previously used this technology to transfer other materials between cells and hope to commercialize the nanoblade for wider use in bioengineering.

Caption: This diagram illustrates the process of transferring mitochondria between cells using the nanoblade technology. Credit: Alexander N. Patananan Courtesy UCLA

Caption: This diagram illustrates the process of transferring mitochondria between cells using the nanoblade technology.
Credit: Alexander N. Patananan Courtesy UCLA

A May 10, 2016 Cell Press news release on EurekAlert, which originated the news item, expands on the theme,

“As a new tool for cell engineering, to truly engineer cells for health purposes and research, I think this is very unique,” says Mike Teitell, a pathologist and bioengineer at the University of California, Los Angeles (UCLA). “We haven’t run into anything so far, up to a few microns in size, that we can’t deliver.”

Teitell and Pei-Yu “Eric” Chiou, also a bioengineer at UCLA, first conceived the idea of a nanoblade several years ago to transfer a nucleus from one cell to another. However, they soon delved into the intersection of stem cell biology and energy metabolism, where the technology could be used to manipulate a cell’s mitochondria. Studying the effects of mutations in the mitochondrial genome, which can cause debilitating or fatal diseases in humans, is tricky for a number of reasons.

“There’s a bottleneck in the field for modifying a cell’s mitochondrial DNA,” says Teitell. “So we are working on a two-step process: edit the mitochondrial genome outside of a cell, and then take those manipulated mitochondria and put them back into the cell. We’re still working on the first step, but we’ve solved that second one quite well.”

The nanoblade apparatus consists of a microscope, laser, and titanium-coated micropipette to act as the “blade,” operated using a joystick controller. When a laser pulse strikes the titanium, the metal heats up, vaporizing the surrounding water layers in the culture media and forming a bubble next to a cell. Within a microsecond, the bubble expands, generating a local force that punctures the cell membrane and creates a passageway several microns long that the “cargo”–in this case, mitochondria–can be pushed through. The cell then rapidly repairs the membrane defect.

Teitell, Chiou, and their team used the nanoblade to insert tagged mitochondria from human breast cancer cells and embryonic kidney cells into cells without mitochondrial DNA. When they sequenced the nuclear and mitochondrial DNA afterwards, the researchers saw that the mitochondria had been successfully transferred and replicated by 2% of the cells, with a range of functionality. Other methods of mitochondrial transfer are hard to control, and when they have been reported to work, the success rates have been only 0.0001%-0.5% according to the researchers.

“The success of the mitochondrial transfer was very encouraging,” says Chiou. “The most exciting application for the nanoblade, to me, is in the study of mitochondria and infectious diseases. This technology brings new capabilities to help advance these fields.”

The team’s aspirations also go well beyond mitochondria, and they’ve already scaled up the nanoblade apparatus into an automated high-throughput version. “We want to make a platform that’s easy to use for everyone and allow researchers to devise anything they can think of a few microns or smaller that would be helpful for their research–whether that’s inserting antibodies, pathogens, synthetic materials, or something else that we haven’t imagined,” says Teitell. “It would be very cool to allow people to do something that they can’t do right now.”

The pipette being used is measured at the microscale but it’s called a nanoblade? Well, perhaps the tip or the edge of the pipette is measured at the nanoscale.

Getting back to the research, here’s a link to and a citation for the paper,

Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells by Ting-Hsiang Wu, Enrico Sagullo, Dana Case, Xin Zheng, Yanjing Li, Jason S. Hong, Tara TeSlaa, Alexander N. Patananan, J. Michael McCaffery, Kayvan Niazi, Daniel Braas, Carla M. Koehler, Thomas G. Graeber, Pei-Yu Chiou, Michael A. Teitell. Cell Metabolism Volume 23, Issue 5, p921–929, 10 May 2016  DOI: http://dx.doi.org/10.1016/j.cmet.2016.04.007

This paper appears to be open access.

A nanoscale bacteria power grid

It’s not often you see the word ‘spectacular’ when reading a science news item but it can be found in an Oct. 21, 2015 news item on ScienceDaily,

Electrical energy from the socket — this convenient type of power supply is apparently used by some microorganisms. Cells can meet their energy needs in the form of electricity through nano-wire connections. Researchers from the Max Planck Institute for Marine Microbiology in Bremen have discovered these possibly smallest power grids in the world when examining cell aggregates of methane degrading microorganisms. They consist of two completely different cell types, which can only jointly degrade methane. Scientists have discovered wire-like connections between the cells, which are relevant in energy exchanges.

It was a spectacular [emphasis mine] scientific finding when researchers discovered electrical wiring between microorganisms using iron as energy source in 2010. Immediately the question came up if electric power exchange is common in other microbially mediated reactions. One of the processes in question was the anaerobic oxidation of methane (AOM) that is responsible for the degradation of the greenhouse gas methane in the seafloor, and therefore has a great relevance for Earth climate. The microorganisms involved have been described for the first time in 2000 by researchers from Bremen and since then have been extensively studied.

This image accompanies the research,

Caption: Electron micrograph of the nanowires shows connecting archaea and sulphate reducing bacteria. The wires stretch out for several micrometres, longer than a single cell. The white bar represents the length of one micrometre. The arrows indicate the nanowires (A=ANME-Archaeen, H=HotSeep-1 partner bacteria). Credit: MPI f. Biophysical Chemistry

Caption: Electron micrograph of the nanowires shows connecting archaea and sulphate reducing bacteria. The wires stretch out for several micrometres, longer than a single cell. The white bar represents the length of one micrometre. The arrows indicate the nanowires (A=ANME-Archaeen, H=HotSeep-1 partner bacteria).
Credit: MPI f. Biophysical Chemistry

A Oct. 21, 2015 Max Planck press release (also on EurekAlert), which originated the news item, provides more information about methane in the ocean, power wires, and electron transporters,

In the ocean, methane is produced from the decay of dead biomass in subsurface sediments. The methane rises upwards to the seafloor, but before reaching the water column it is degraded by special consortia of archaea and bacteria. The archaea take up methane and oxidise it to carbonate. They pass on energy to their partner bacteria, so that the reaction can proceed. The bacteria respire sulphate instead of oxygen to gain energy (sulphate reducers). This may be an ancient metabolism, already relevant billions of years ago when the Earth’s atmosphere was oxygen-free. Yet today it remains unknown how the anaerobic oxidation of methane works biochemically.

Gunter Wegener, who authors the publication together with PhD student Viola Krukenberg, says: “We focused on thermophilic AOM consortia living at 60 degrees Celsius. For the first time we were able to isolate the partner bacteria to grow them alone. Then we systematically compared the physiology of the isolate with that of the AOM culture. We wanted to know which substances can serve as an energy carrier between the archaea and sulphate reducers.” Most compounds were ruled out quickly. At first, hydrogen was considered as energy source. However, the archaea did not produce sufficient hydrogen to explain the growth of sulphate reducers – hence the researchers had to change their strategy.

Direct power wires and electron transporters

One possible alternative was to look for direct connections channelling electrons between the cells. Using electron microscopy on the thermophilic AOM cultures this idea was confirmed. Dietmar Riedel, head of electron microscopy facilities at the Max Planck Institute in Goettingen says: “It was really challenging to visualize the cable-like structures. We embedded aggregates under high pressure using different embedding media. Ultrathin sections of these aggregates were then examined in near-native state using transmission electron microscopy.”

Viola Krukenberg adds: “We found all genes necessary for biosynthesis of the cellular connections called pili. Only when methane is added as energy source these genes are activated and pili are formed between bacteria and archaea.”

With length of several micrometres the wires can exceed the length of the cells by far, but their diameter is only a few nanometres. These wires provide the contact between the closely spaced cells and explain the spatial structure of the consortium, as was shown by a team of researchers led by Victoria Orphan from Caltech.

“Consortia of archaea and bacteria are abundant in nature. Our next step is to see whether other types also show such nanowire-like connections. It is important to understand how methane-degrading microbial consortia work, as they provide important functions in nature”, explains Antje Boetius, leader of the research group at the Institute in Bremen.

Here’s a link to and a citation for the paper,

Intercellular wiring enables electron transfer between methanotrophic archaea and bacteria by Gunter Wegener, Viola Krukenberg, Dietmar Riedel, Halina E. Tegetmeyer, & Antje Boetius. Nature 526, 587–590 (22 October 2015) doi:10.1038/nature15733 Published online 21 October 2015

This paper is behind a paywall.