Joel P Joseph Biological Sciences, Health, Medicine, News

Using viruses to kill Tuberculosis bacteria

This article was first published in Research Matters. Read the article, as it appeared on researchmatters.in, here.

Viruses are infamous for the infectious diseases they cause in different organisms — the year 2020 has proved it for us to see. But, a virus that causes an infection in one organism could be harmless in another. The Nipah virus, for example, is harmless in bats but causes a deadly disease in humans. Likewise, there are a group of viruses called bacteriophages that infect and kill bacteria but are harmless in humans. Within this group of viruses are myriad individuals, each one specific to certain bacteria.

In a recent study, researchers from the Indian Institute of Science (IISc), Bengaluru, have found that a cocktail of bacteriophages could kill Mycobacterium tuberculosis – the bacteria that causes Tuberculosis (TB), and its cousin Mycobacterium smegmatis­. The study was led by Rachit Agarwal, Assistant Professor at the Centre for BioSystems Science and Engineering, IISc, and the findings were published in the journal Frontiers in Microbiology.

Tuberculosis (TB) is one of the top 10 causes of death worldwide. It affected 10 million people and killed 1.4 million of them last year. India has the highest burden of TB worldwide, with nearly 4.5 lakh deathsreported in 2018. When M. tuberculosis infects a person, their immune system launches an attack against it. A group of immune cells, called macrophages, engulf the bacteria to form a packet inside the cell containing the bacteria with a slightly acidic environment. Typically, this packet would fuse with another component inside the cell, called the lysosome, which would make the environment more acidic and thereby kill the bacteria.

However, M. tuberculosis and its relatives have a smart way of escaping this process. They not only block this process of creating a more acidic environment, but they also thrive in an acidic environment and in low-oxygen conditions where other cells would die! They switch to a state in which they do not multiply fast, but grow slowly, residing inside these acidic compartments made by the body’s immune system.

Doctors treat tuberculosis with a combination of drugs that includes antibiotics. Over the years, the indiscriminate use of antibiotics has led the bacteria to develop resistance to these drugs, resulting in antibiotic-resistant tuberculosis infection. India also has the highest burden of such infections.

“The main motivation behind our study was the fact that antibiotic-resistance has been on the rise and is predicted to be a major global health crisis soon,” says Yeswanth C Kalapala, the lead author of the study.

In recent years, researchers have explored the use of bacteriophages against tuberculosis bacteria to curb their growth and kill them. The current study is no different. The researchers have studied how bacteriophages work against Mycobacterium in various disease-mimicking environments.

“We found that these bacteriophages were effective against Mycobacterium in various disease-mimicking conditions like acidic environment, low oxygen concentration and nutrient starvation,” says Rachit.

The researchers first studied the effect of single bacteriophage on the growth of Mycobacterium and later used a mixture of them — five different bacteriophages against M. smegmatis, and three against M. tuberculosis — in their lab.

“The bacteria develop some tolerance against individual phages over time, but a cocktail of phages inhibit the growth of the bacteria for a longer time and delay the development of tolerance to phages,” explains Pallavi R Sharma, one of the authors of the study.

The researchers found that the cocktail was effective in acidic environments, low-oxygen and low-nutrition conditions — all of which are present in cells infected with tuberculosis. It was also working against slow-growing bacteria. They then used the cocktail in combination with rifampicin, an antibiotic conventionally used to treat TB, on lab-grown bacteria. They found that the combination had a synergistic effect in reducing the growth when compared to the using either one separately.

As TB cases caused by antibiotic-resistant bacteria are on the rise, the authors also looked into the effect of the phage cocktail on an antibiotic-resistant strain of M. smegmatis. M. smegmatis generally do not cause a disease, but behave similar to other Mycobacteria. This allows researchers to use this bacteria in laboratory conditions where safety requirements are lesser than those for the use of M. tuberculosis, while giving them an idea of how M. tuberculosis might behave in similar conditions. Besides, M. smegmatis reproduce faster than M. tuberculosis. So, the authors used the antibiotic resistant M. smegmatis as a model to study how other antibiotic resistant Mycobacteria react to phage cocktails.

“We found that the five-phage cocktail was effective in infecting and killing antibiotic-resistant M. smegmatis. We also saw that the phage cocktail complemented rifampicin and eliminated the bacteria that were resistant to it,” says Yeswanth.

Following these interesting observations, the researchers are planning to study the effect of these phage cocktails on Mycobacterium tuberculosis growing inside human cells cultured in the lab and animal models like mice.

“We wish to see how this therapy can be used in animals and later translated to humans to treat TB, particularly in the case of drug-resistant TB,” signs off Rachit.

Joel P Joseph News, Science & Technology

Bengaluru based start-up designs an anti-touch band

Grasp bionics, a Bengaluru-based start-up has devised an anti-touch band. The band stops people from accidentally touching the face.

Anti-touch band packed. Picture Courtesy: Grasp Bionics
Anti-touch Band. Picture Courtesy: Grasp Bionics

As of today, there are 37916 active cases of COVID-19 in India and the epidemic has claimed 1886 lives. The increase in the number of cases led the government to declare a nationwide lockdown, which has now been extended for the third time. While these restrictions are in place, scientists and innovators are striving hard to fight the epidemic. With no vaccine or drug currently available, one can only take precautionary measures like physical distancing, restricted movement, hand hygiene and wearing masks.

A study shows that, on average, we touch our face about 23 times in an hour. And we know that a person can get infected with the virus if one touches the face after touching an infected surface. So, as we come out of this lockdown, touching the face can now be riskier than ever.

group photo-01
Team Grasp Bionics. L to R: Nilesh Walke, Vinay V, Arvind Sahu, Varsha, Abhijith R

Grasp Bionics, a Bengaluru based start-up, has devised an interesting solution to this problem. “We observed that however cautious we are, we tend to touch our nose and mouth. When it is intentional, we may do it after washing our hands. But most of the time it is accidental.” Vinay V, Co-founder & Director of Grasp Bionics, said. “We thought that a solution for this could be of huge benefit during the removal of lockdown. That is how we came up with this anti-touch band.”

The band restricts the movement about the elbow thereby the touch on the face. It is made of cloth and costs about 100 rupees apiece. The team is working with local tailors for mass production as it could also be a source of income for the tailors who may not have regular work during the lockdown and phased relaxation.

“We plan to make the band available for purchase in general stores & medical stores,” Vinay said. The team is looking for distributors, who can make their product available to the customers. They are also working on making the design open-source so that people can make DIY anti-touch bands.

Grasp Bionics is a MedTech company that builds bionic devices like prosthetic arms. Their flagship product PURAK is a wearable prosthetic limb that provides better control and sense for those who have lost their arms. The team was a winner of Elevate 2019, a program organized by Department of IT & BT, Government of Karnataka, to identify and fund 100 start-ups with innovative ideas.

Here is a video of how the band works. Place your order for the band here.

Joel P Joseph Biological Sciences, News

Scientists discover that structures inside mitochondria reshape continuously in living cells

Scientists from Heinrich Heine University Düsseldorf and University of California Los Angeles recently discovered that cristae – structures formed by the inner covering of mitochondria – keep remodelling continuously. The team led by Prof. Andreas S. Reichert made this discovery by seeing live cells using a high-end technique in microscopy. This technique, called stimulated emission depletion (STED) super-resolution nanoscopy, enables one to see details of an organelle in the cell – of the size of 50 nm (about 1000 times smaller than a speck of dust). The study was published in the journal EMBO Reports.     

CJ dyna,ics

STED nanoscopy images showing cristae remodelling 

Mitochondria are organelles in the cell popularly known as the powerhouse of the cell. They form a dynamic network, change their shape, fuse or split. As for their structure, mitochondria have two membranes covering them – the outer membrane and the inner membrane. The inner membrane folds inward to form structures called cristae (sing. crista), on which many important proteins reside. These proteins include ATP synthase – the machine that makes ATP, the energy currency of the cell. Curved, circular or pore-like structures called crista junctions separate the cristae and the rest of the inner membrane.

Other scientists have shown that abnormal or altered cristae in diseases like cancer, diabetes and neurodegeneration. In normal conditions, however, cristae are thought to be static. Scientists from Heinrich Heine University Düsseldorf and University of California Los Angeles led by Prof. Reichert have now found that the cristae membrane and crista junctions reshape continuously. They also found that a protein complex called the mitochondrial contact site and cristae organizing system (MICOS) orchestrate these remodelling events.

 “Prof. Reichert’s lab has been working on the mechanisms of cristae remodelling. We were working on the function of MICOS complex located at the crista junctions,” Dr Arun Kumar Kondadi, one of the first authors of the paper, said. But one could never investigate if the cristae were dynamic or not as there was no technique that enabled researchers to see the details inside an organelle. “For more than half a century, it was rather assumed that cristae are static,” Dr Kondadi said. “Super-resolution microscopy techniques paved us the way to discover something really fascinating.”

In this study, using STED super-resolution nanoscopy, researchers have shown that adjacent crista junctions come together and separate from each other reversibly in human cells. For this, the researchers stained cristae membranes using dyes (or marked proteins present on the inner membrane of mitochondria) and observed how the cristae remodel using STED super-resolution nanoscopy. These experiments showed that cristae undergo membrane remodelling continuously.

According to Prof. Reichert, as quoted in the Press Release from Heinrich Heine University, the observations of this study lead us to a new model – Cristae Fission–Fusion (CriFF) model – that cristae can stay as isolated vesicles within mitochondria and then re-fuse with the inner membrane after fission. The study shows that this fusion is carried out by the MICOS complex. MIC60 – one of the proteins of the complex – initiates the process and marks the crista junction. It then recruits other proteins of the MICOS complex (MIC10, MIC13, MIC19, MIC25, MIC26 and MIC27) and completes the assembly. These crista junctions now become the sites from where cristae form. These junctions keep merging and splitting. The cristae could, therefore, pinch off from these junctions and come back to the same junction or another.

This study has opened a new field in research where one could investigate how a mitochondrion ensures quality control within itself. For all we know now, remodelling and reshaping of mitochondria in a cell can control the fate of the cell. This study has gone one step ahead to show how structures within the mitochondrial remodel continuously. How these events inside mitochondria can affect the mitochondria, and how this impact on mitochondria can influence the cell will be interesting to study.

Read the full paper here

Image Courtesy & Article Source: EMBO Reports

Joel P Joseph Biological Sciences, Medicine, News

Scientists identify a compound that protects neurons from degeneration

460711949-56a793d25f9b58b7d0ebda5c     neuron

Scientists from the Bengaluru-based institution Jawaharlal Nehru Center for Advanced Scientific Research (JNCASR), with the help of National Institute of Mental Health and Allied Sciences (NIMHANS) and Center for Cellular and Molecular Platforms (C-CAMP), have identified a molecule that could clear toxic aggregates in the brain and reduce the loss of neurons (neurodegeneration). The study was led by Dr Ravi Manjithaya, Associate Professor, Autophagy laboratory, Molecular Biology and Genetics Unit, and Associate Faculty of Neuroscience, JNCASR. The findings were recently published by the Lancet journal EBioMedicine.

Parkinson’s disease is a neurodegenerative disease that affects 10 million people worldwide. In Parkinson’s disease, misfolded proteins form clumps in a set of neurons in the brain – the neurons that release dopamine (dopaminergic neurons). These aggregates, known as Lewy bodies, majorly constitute a misfolded protein called α-synuclein and result in the loss of dopaminergic neurons. Loss of dopaminergic neurons affects the movement and cognition of a person. The person is unable to walk properly, has shaky hands and legs, and becomes forgetful, drastically affecting his/her quality of life.

Several scientists across the globe have tried to develop drugs that reverse this situation but have failed. In this study, the team of researchers identified a chemical compound that blocks a protein (c-abl kinase), thereby clearing the toxic aggregates formed. The team had to screen numerous small molecules before pinning down on the small molecule, PD180970.

The team overexpressed (forced synthesis beyond natural limit in the cell) α-synuclein in yeast cells, making it toxic for the cells and killing them. This model could mimic how α-synuclein aggregates kill neurons and the researchers tested the small molecules on this. “We screened a small molecule library and identified those that rescued growth of yeast cells expressing the Parkinson’s disease-associated protein, α-synuclein,” Dr Manjithaya said. “Later we narrowed down to molecules that were rescuing the growth defect in an autophagy-dependent manner.”

The team found that PD180970 could inhibit c-abl kinase and induce autophagy to reduce the toxicity in neurons caused by the aggregates. This was evidenced by their studies on neuronal cell lines and in mouse midbrain. In addition to clearing aggregates, preventing the inflammation of neurons is important to stop neurodegeneration. “A small molecule that induces autophagy to clear the aggregates and also has anti-inflammatory property would be more potent in curbing neurodegeneration,” Dr Manjithaya said. “This is how we think PD180970 works,” he added.

The collaborations with Dr Phalguni Alladi, Senior Scientific Officer, NIMHANS, Dr James Clement Chelliah, Neuroscience Unit, JNCASR, and Dr Taslimarif Saiyed, CEO and Director, C-CAMP, were vital in this aspect. “While my lab focuses on the autophagy aspects, my collaborator Phalguni Alladi at NIMHANS mentioned about the impact of neuroinflammation. To this end, we collaborated with Taslim’s lab at C-CAMP to test if any of our molecules had anti-inflammatory properties.” Dr Manjithaya said. “PD180970 was the most effective among the ones they tested.” PD180970 prevented neuroinflammation by inhibiting cytokines like IL-6 (interleukin-6) and MCP-1 (monocyte chemoattractant protein-1), which facilitate inflammation.

Dr Manjithaya is highly appreciative of collaborations in science. “As a young researcher, having collaborations is looked down upon in our country although it is the norm and is encouraged in the western world,” he said. “Good collaborations bring in various aspects that make a study complete. Apart from their unique expertise and direction, there is further confirmation of the work, and more importantly different – sometimes contrasting – views. That makes you think more critically about your data and gives a fresh perspective. This study is a culmination of efforts from four labs across three institutions.”

In mice where the dopaminergic neurons were degenerated by injecting a chemical called MPTP. Treatment with PD180970 along with MPTP injection improved the condition. This was supported by evidence from the changes at a molecular level in the brain and the behavioural changes that occurred in the mice.

That’s not all. The lab is working with Vipragen Bioscience to patent the molecule and take it forward to the next stage in drug development.

Read the full paper here

Pictures from verywellmind.com andeinstein.yu.edu

Joel P Joseph News, Science Outreach

A group of 20-somethings are changing the way biology is taught at school

IMG-20191005-WA0021   IMG_1061

Chennai: Cambrionics Life Science – a community of biologists based in Chennai – is changing the way biology is taught in schools. The enterprise founded by five 20-somethings has reached over 3500 students in Tamil Nadu with their flagship program, Teaching Alternate Biological Science (T.A.B.S). T.A.B.S is a 12-week experimental learning program in Life sciences for school kids in grades 6 to 9.

The program is built on a research-based learning framework, where students spend four weeks experimenting with each model organism. The course includes model systems like Zebrafish or Drosophila, Microbes, and Plants. Through these experiments, students are exposed to various themes in biology including neurobiology, genetics, forensic biology, microbiology, developmental biology, cell biology, toxicology, astrobiology, hydroponics, aquaponics and farming.

The program is designed at three levels – beginner, intermediate and advanced, each level providing varying degrees of exposure to biology. While the beginner level is designed to inspire students and ignite a passion for biology, the intermediate level is aimed to develop critical thinking and the advanced level exposes them to various career opportunities in biology and encourages them to develop scientific solutions to a given problem.

The upcoming T.A.B.S program starts on Saturday, November 30, 2019. The 12-week program is conducted every Saturday from 4:00 PM – 6:00 PM at KK Nagar, Chennai.

To register for this module of T.A.B.S, contact the team at +91-7845133745 or register here. You could also contribute to the cause here.

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 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