Joel P Joseph Biological Sciences, News, Science & Technology

NOW YOU SEE ME: First Cryo-EM image of New World Hantaviruses

Scientists from the USA recently published the first Cryo-EM image of New World Hantaviruses. The team, led by Colleen B. Jonsson, obtained the Cryo-EM images of three New World Hantaviruses – Andes Virus (ANDV), Sin Nombre Virus (SNV), and Black Creek Canal Virus (BCCV). The study was published in the journal Viruses.     

  SNV      Capture

Left: Cryo-EM image of Sin Nombre Virus (SNV). Right: Members of the research team. L to R: Mariah Taylor, Amar Parvate, Colleen B. Jonsson

The team of seven researchers employed cryo-electron microscopy (cryo-EM) to understand the structural features a group of viruses called Hantaviruses. The technique helped them unravel the features of two types of Hantaviruses – Old World Hantavirus and New World Hantaviruses. While the former is prevalent in Eurasia, the latter is found in the Americas. Old World Hantaviruses include the viruses that cause haemorrhagic fever with renal syndrome (HFRS), whereas New World hantaviruses include those viruses that cause hantavirus pulmonary syndrome (HPS).

“I had not intended to work on these structures from the onset,” Amar Parvate, the first author of the paper says. “The idea was to investigate the spread of hantaviruses through cells using Transmission Electron Microscopy (TEM). But I realized that these viruses were not safe. We had to work on them in a highly contained environment (BSL3). There was no way to safely load them on a cryo-EM.” The team then worked on a method to inactive these viruses, without compromising their quality, and load them safely to see how they look through the cryo-EM. It took Amar and his collaborators three years to optimize the method on a prototype virus. They then applied it to several BSL3 hantaviruses the results of which are published in the paper.

The cryo-EM images of Andes Virus (ANDV), Sin Nombre Virus (SNV), and Black Creek Canal Virus (BCCV) published by this team are the first cryo-EM images of New World Hantaviruses. These images have revealed diverse features and sizes of New World viruses. They are round, tubular or irregular. While BCCV were mostly tubular, SNV were mostly irregular.

“Most of the Hantavirus community was looking at Old World Hantavirus morphology and assuming that the virions are all round. One reason was that they could not put these viruses on a cryo-EM was the containment restriction I mentioned earlier. Even after I had the initial results, other researchers were sceptical if the viruses were inactivated or whether the method itself distorted the morphology. My images proved that there are more diverse features to these viruses rather than just being round” Amar explained. “The most striking finding was the long tube-like morphology of one of the New World Hantaviruses.”

Amar’s method is now available to other researchers working on these kinds of viruses and paves the way to further discoveries in viral studies. “I am hoping other groups use my method to finally tease out structural details of other dangerous (BSL3/4) viruses that had been recalcitrant cryo-EM and structural studies,” Amar said.

Of course, the team had to do some things very differently to achieve this. Electron microscopists have traditionally fixed their room temperature biological samples on the grids using glutaraldehyde. To get an image of higher resolution using cryo-EM, they fixed the samples with a very mild fixation technique using glutaraldehyde. Amar and colleagues combined the two techniques into one. This was the thought that brought the breakthrough.

Amar is excited about the possibilities that his work has opened to researchers studying viruses. “Currently, there are very few cryo-EM facilities in the world that can handle BSL3 samples. Although there are advances being made in this direction, most highly contained (BSL3) labs do not have access to cryo-EM. My method proposes a way to use cryo-EM outside the containment for any BSL3 viruses” Amar said. “Once the virus is inactivated, it can be safely taken out and even shipped to a completely different institute for cryo-EM analysis. This type of extension of my method may eventually help us analyse morphologies of multiple viruses for which the most we have now are one or two TEM images collected in the 1980s.”

The implications of this research, however, do not end there. All these findings will ultimately lead to the greater goal of developing drugs and vaccines to fight these viruses. But those will come only later – when we have a better understanding of the structural features of the viruses. For all we know now, the images published by the group and the method that they have put has given the scientific community a great stride in studying dangerous viruses.

Joel P Joseph Biological Sciences, News

Scientists discover a new gene essential for hearing

Scientists have associated a new gene – Clrn2 – with hearing in mammals. Led by Amraoui and Bowl, the study was a collaborative effort of 32 researchers from institutes in the UK, France, and the USA. The findings were recently published in the journal EMBO Molecular Medicine.

clrn2

Cilia bundle in the inner ear. Picture adopted from EMBO Molecular Medicine

The sense of hearing results from a combination of events of physical and biological sciences. Mechanical energy from sound waves falling on the inner ear must be converted to neuronal signals for a person to hear. The process is taken care of by the specialized hair cells in the inner ear. On the tip of each hair cell is a bundle of cilia, some tall and some short, arranged in a specific manner and tethered to a complex. The movement of the inner ear fluid, caused by sound, deflects these hair cell bundles towards the tallest cilia. The tension created in the tip open the channels in the complex attached to it. The complex releases neuronal signals, completing the conversion of mechanical energy to neuronal signals.

Hearing loss can be caused by environmental factors, genetic factors or a combination of both. Although scientists have managed to understand early-onset hearing loss and hereditary hearing loss to an extent, very little is known about the genetics behind age-related hearing loss. The research conducted by this team has implicated the involvement of Clrn2 in age-related or progressive hearing loss.

The team mutated the Clrn2 gene in mouse and investigated its effect only to discover a progressive hearing loss. To check if the finding applied to humans as well, the group analysed the CLRN2 gene sequences of 5 lakh people from the UK biobank participants data. Data was segregated as hearing loss cases (163, 333) and controls with normal hearing ability (102,832). The cases and controls selected were above 50 years of age. The classification was made based on the participants’ response two questions recorded in the published paper – (i) Do you have any difficulty with your hearing? (ii) Do you find it difficult to follow a conversation if there is background noise (such as TV, radio, children playing)?  Those who answered ‘No’ to both were controls and other were cases. On analysing their CLRN2 gene sequence, the group found that those who had difficulty in hearing harboured mutations in their CLRN2 genes.

With further experiments on mice, the team discovered that while Clrn2 was necessary for the maintenance of the bundle of cilia in the hair cells of the inner ear after they had been formed. Mutations in this gene would, therefore, lead to poor maintenance of the bundle leading to progressive hearing loss.

From this study, scientists and clinicians now have a reference point to test for hearing loss because of ageing. We may be looking at days when the auditory tests, which are subjective, are replaced by the objective genetic tests for deafness.

Joel P Joseph Interviews, Science & Technology

High resolution Cryo-EM structure from the recent national facility at Bangalore

Scientists at the Bengaluru-based Institute of Stem Cell Biology and Regenerative Medicine (InStem) and National Centre for Biological Sciences (NCBS) have unravelled the structure of a bacterial enzyme, PaaZ, using cryo-electron microscopy. The study was conducted in collaboration with the MRC Laboratory of Molecular Biology, Cambridge, UK and Carver College of Medicine, University of Iowa, USA. The structure was recently published in the journal Nature Communications.

This is the latest high resolution structure (less than 3A) to be published from the recently established state-of-the-art single electron Cryo-EM facility at Bangalore Life Science Cluster.

Here is an exclusive interview with Nitish Sathyanarayanan, one of the first authors of the paper, on this incredible journey.

PaaZ2.png     PaaZ image

1. What got you interested in structural biology?

I began to get fascinated by structural biology during my Bachelor’s degree, where I spent a lot of time reading Lehninger “Principles of Biochemistry”. If you notice keenly, almost every section of this bible contains a structural explanation. It was this interest in biochemistry and enzymology that got me into the field of structural biology. Since I come from Biology training (not Physics or Chemistry), I have always used structural biology as a set of ‘tools/techniques’ to understand the functions of enzymes.

2. How did you come to study the structure of PaaZ?

We stumbled upon PaaZ due to our interest in multi-domain proteins which are involved in aromatic ring degradation. Phenyl Acetate (Paa) degradation pathway is also called “hybrid pathway” since its mechanism of ring degradation has features of both aerobic and anaerobic pathways. Our specific interest was to understand the functioning of this enzyme through its structure and biochemistry. It is also important since several environmental pollutants such as styrene converge to Paa through peripheral pathways.

3. What were the challenges you faced in the project?

We were essentially trying to understand how the enzyme functioned using its structure. We spent nearly 18-24 months crystallizing the protein, with no success in obtaining a diffraction quality crystal. We then attempted an integrated structure modelling approach (Light scattering, SAXS, MD and Modelling) to obtain a model with limited success. This was also the time when structural biology domain was undergoing “resolution revolution” (the term used to describe new advances in Cryo-EM). Since the protein was big and we had limited success with other methods, we decided to explore Cryo-EM to understand the structure.

4. What was your reaction when you first solved these structures?

I was amazed! Here was a protein that I purified for several years. I had only imagined it as a liquid or a blue band on an SDS-PAGE gel or as a gel filtration profile. I never knew how it looked. It was like a blind date. When you talk to someone only over the phone for several years, you build your own imagination. It was similar. But when I saw the structure as a Cryo-EM map, it was more beautiful than I ever imagined.

5. How do you plan on taking the findings of this project further as a scientist and entrepreneur?

For me, PhD was “one big project” since I was involved with multiple projects and simultaneously part of three labs (Bioinformatics, Structural biology and Cryo-EM). As my mentor Rams (Prof. S. Ramaswamy) always said, PhD is training – training an individual to ask tough questions, design an experiment to answer these questions, perform and reproduce an experiment, and find a conclusion. In between these four steps, a PhD student often goes through innumerable failed experiments. It is the ability to break a complex problem into simpler tasks, find answers to these smaller tasks (with several failures) and, in the end, put all pieces of the puzzle to build a complete picture. This is a life lesson I plan to leverage in my next journey as a tech entrepreneur.

6. Your message to academic peers struggling in their PhD?

If you are not struggling, then there is a problem. It’s called “re-search” for a reason. I feel that one should have read at least about a hundred papers in that area of research in the first 6 months. My humble suggestion is to spend a considerable amount of time reading and staying updated with literature. This can be a great source of new ideas. The other benefit is that you will become aware of the labs that work in a similar field. You could always request for plasmids, protocols, reagents, suggestions etc. from them. If you do not read enough, you cannot do any of this. And please remember, by the end of your PhD, you should have become an expert in that field.

Picture adapted from the published paper.

Link to the full-text of the article: https://www.nature.com/articles/s41467-019-11931-1

Joel P Joseph Biological Sciences, Medicine, News

Cancer drugs may not be working the way we think they work

Scientists at Cold Spring Harbor Laboratory (CSHL) have discovered that many anti-cancer drugs do not work the way we think they work. The study, led by Jason M. Sheltzer, evaluated 10 anti-cancer drugs in the pre-clinical or clinical trials. Findings of this research may help in establishing more stringent tests for drugs before trying them on humans.

cancer colorful word in the wooden background

Sheltzer’s earlier investigations of a protein, MELK, paved way to this study. Several reports had suggested that MELK was essential for the survival of cancer cells. But Sheltzer found otherwise – cancer cells survived even in the absence of MELK. They also found that an anti-cancer drug (OTS167), which the clinicians claimed would target MELK, killed cancer cells that were depleted of MELK. Clearly, OTS167 did not target MELK to kill the cancer cells.

Sheltzer adopted this strategy to study more anti-cancer drugs. The team shortlisted 10 anti-cancer drugs that were in pre-clinical or clinical testing. They selected drugs that clinicians claimed would stop the proliferation of cancer cells by targeting a single protein. The researchers then deleted the gene that coded for the claimed target protein using the CRISPR-Cas9 technology and checked if the cancer cells survived. But cancer cells were killed. This suggested that the drugs were not targeting these proteins.

The researchers particularly studied a drug (OTS964) targeting the protein PBK in greater detail. They gave enough time for cancer cells to accumulate mutations and develop resistance to OTS964 to find what protein the drug targeted. Cancer cells are genetically unstable and accumulate mutations over time. These mutations, resulting in changes in the protein they code for, restrain the drug from binding to the target protein. Knowing the genes that are mutated help us understand the actual target proteins. On analysis, they found that the gene coding for CDK11 had several mutations in it. This indicated that CDK11 was the actual target of this drug.

This may be one reason why 97 per cent of cancer drugs tested in clinical trials do not go on to receive US FDA approval. Understanding the accurate ‘mode of action’ of a drug increases the chances of its success. It is possible that the earlier experimental methods of inhibiting a gene or protein – RNAi and small molecule inhibitors – could be blocking some other protein in the cell. This may well the reason why drugs work differently than how we thought they would.

With advanced technologies like CRISPR-Cas9 technology, we can be more accurate. As the current study showed, this can be used as a method to test the claimed “mode of action” of the drug before they are tested on humans. This study also gives us a method to understand what proteins or genes are essential for the survival of cancer cells.

Pic Courtesy: China Daily