Detection of mercury and discrimination of breast cancerous and non-cancerous cells

Mr. Subhajit Chakraborty’s interview with Bio Patrika hosting “Vigyaan Patrika”, a series of author interviews. Subhajit is from West Bengal, India. He completed his B.Sc. in Chemistry from Ramakrishna Mission Vivekananda Centenary College, Rahara (2013). He did M.Sc. from the Presidency University, Kolkata in 2015. He joined the Ph.D. program (2016) in the Department of Chemistry of the Indian Institute of Science Education and Research Bhopal, under the supervision of Professor Saptarshi Mukherjee. His research work is mainly focused on the Fluorescence spectroscopy and development of luminescent metal nanomaterials to study their characteristic photophysical and morphological aspects and their applications in the various interdisciplinary field of sciences. Here, Subhajit talks about his work on ‘Protein-Templated Gold Nanoclusters as Specific Bio-Imaging Probe for the Detection of Hg(II) Ions in In Vivo and In Vitro Systems: Discriminating MDA-MB-231 and MCF10A Cells’ published in Analyst.

Listen audio

How would you explain your paper’s key results to the non-scientific community?

Mercury (Hg) is a global poison for ecological systems particularly humans. Artisanal and small-scale gold mining (ASGM) is one of the main sources of mercury pollution after anthropogenic mercury emissions. The most dangerous aspect is that it can affect the central nervous system along with triggering other problems like hyper-irritability, fatigue, behavioral changes, hallucinations, etc. Mercury in its ionic form can readily combine with base pairs of DNA causing maximum damage to the endothelial cells, and cardiovascular functions. Additionally, it is expected that mercury will bind with thiol-containing biomolecules when present inside the body and can damper the normal metabolic activities of these bio-molecules. Thus, the highly sensitive detection of Hg(II) is very crucial (very low Limit of Detection (LOD)) and always a challenging task. Fluorophores that have prominent selective detection ability, have robust nature towards external physical and chemical perturbations, and can be used for single molecular investigations are termed as good sensors. They also have optimum quantum yields as well as very good photostability. Noble metal nanoclusters (NCs) templated by protein motifs have gained more impact of late as they render additional stability to the cluster core and thus generate interesting optical properties.

Schematic

Highlighting these characteristics, we have carried out the facile and optimized synthesis of gold nanocluster (AuNCs) within a protein (human serum albumin, HSA) template. Most interestingly, these AuNCs possess remarkable photo-, thermal- and core-cluster stability for more than a year (unaltered luminescence properties). We have characterized as-synthesized AuNCs using various spectroscopic and microscopic experiments. The core of these AuNCs consists of 25 gold atoms, which have been estimated by both the experimental and theoretical approaches. Importantly, we conclusively demonstrated the application of AuNCs as robust and photo-stable single molecular probes with the capability to function as highly sensitive (sub-nanomolar detection limit) and selective Hg(II) sensors. Our NCs proved to be quite beneficial in detecting the crucial and toxic Hg ions in solution as well as within a biomolecule. By artificially fabricating an Insulin-Hg complex, our AuNCs were able to efficiently estimate the bound Hg(II) under physiological conditions, thus emphasizing that the detection ability was conserved even at the single molecular resolution. Further study reveals that our AuNCs are also specific for intercellular localization. It specifically endocytoses inside the cancerous cell lines (MDA-MB-231) as revealed by the cell imaging study. Another important feature of this study is that these AuNCs also have the ability to detect Hg(II) ions inside the cell. The fluorescence of AuNCs was quenched in presence of Hg(II) ion inside the MDA-MB-231 cell lines. Thus, the output of our study apprises the sensitive in vivo as well as in vitro detection of Hg(II) ions using AuNCs as a probe.

The interesting feature of this work is that our AuNCs can detect the Hg(II) ions when it is bound with insulin biomolecule.

What are the possible consequences of these findings for your research area?

In this investigation, we have successfully developed intense red luminescent highly stable gold nanoclusters (AuNCs). These AuNCs demonstrated very efficient detection ability towards the Hg(II) ions by quenching their luminescence emission intensity. It is expected that the alteration and modification of the (S-S) bonds of insulin can occur in presence of Hg(II) ions. Our single molecular spectroscopic studies reveal that this detection limit can be extended up to the sub-nanomolar regime. The FCS analysis helped us to achieve very low LOD for Hg(II) detection (~0.01 nM) which is appreciably lower than the international standards set for Hg(II) levels in drinking water (10 nM) according to the U.S. EPA. The interesting feature of this work is that our AuNCs can detect the Hg(II) ions when it is bound with insulin biomolecule. This report shows another important feature of this investigation that is the specificity towards encapsulation of these AuNCs inside the triple-negative breast cancer (TNBC) cell, MDA-MB-231 in comparison to normal cell lines, MCF10A, and ability of detection of Hg(II) ions inside the MDA-MB-231 cell lines.

In a brief, this report can provide a very stable nanomaterial that can sensitively detect Hg(II) in in vitro as well as in vivo environments.

What was the exciting moment (eureka moment) during your research?

The overall journey of this work was exciting to me as each new experiment led to a new set of data, which in turn made the overall study more interesting and application-oriented. The preparation of the AuNCs and the observation of its intense luminescence had generated the initial excitement. The data quality, analysis, and correlation of all experimental techniques are always very satisfying at the end of the day for any researcher and I was no exception. Additionally, the concept that our work can be extended for in vivo cell imaging thereby sensing of Hg(II) inside the cell lines added color to the excitement.

What do you hope to do next?

As these AuNCs can detect Hg(II) ions in various systems, we can use this to detect mercury in drinking water and can thereby analyze the quality of the water. In another aspect, the biomolecular dynamics inside the cell are always important to investigate. We are planning to use these AuNCs as a bio-marker and will try to investigate the inter-cellular environments. We shall also monitor the dynamics of various bio-molecules inside the cells by utilizing FCS and FLIM analyses.

Where do you seek scientific inspiration?

I always feel investigating the unrevealed facts and knowing the unknown inspires me a lot. The research environment in my lab and more importantly, the inspiration from my thesis supervisor Professor Saptarshi Mukherjee all the time (and even today) helps me to move ahead every day. New research reports in various journals, handling new instruments, analysis of new data, etc. definitely creates interest in me and motivates me to work and explore new(er) aspects of science.

How do you intend to help Indian science improve?

Indian science has already evolved to a great extent, still more improvements are needed. According to me, the interest in science and intention of scientific investigation can be created from the school level of education. Another important factor is the development of genuine interest in the understanding of new knowledge inside and outside the laboratory. If I get a chance, then I would like to focus on my further research by creating a group to understand in-depth fundamental aspects of the science along with the results which can be applied to our society. There should be a balance between fundamental and applied research. We need good teachers at all strata so that the thirst among students always remains and they can ask and address the correct questions which will take care of the societal needs. Today Indian science needs a proper synergy between education and research.

Reference

S. Chakraborty, A. Nandy, S. Ghosh, N. K. Das, S. Parveen, S. Datta and S. Mukherjee, Protein-Templated Gold Nanoclusters as Specific Bio-Imaging Probe for the Detection of Hg(II) Ions in In Vivo and In Vitro Systems: Discriminating MDA-MB-231 and MCF10A Cells. Analyst 2021, 146, 1455-1463.

Email: subhajit16@iiserb.ac.in, saptarshi@iiserb.ac.in

Edited by: Ritvi Shah

Job opening in Dr.Reddys

We are Hiring research Associates – Formulation and Drug Product Development for Dr.Reddys, Biologics, Hyderabad.
Job Description : Design and conduct process development studies for the conversion of DS to DP,
Performing drug product formulation activities,
Screening and selection of container closures,
Performing stability studies,
Performing process characterization studies,
Able to plan and execute independently,
Statistical skills would add on value for the position.
Mandatory Skills :
Knowledge on Biosimilars development, Drug Product Process Development Studies (including knowledge on filters, tubings, hold time studies, etc), Formulation studies, Container closure screening, stability studies (analytics), documentation

Applications & References can be sent Across ”Nareshkumar.g@drreddys.com” 

#formulationdevelopment #biosimilars

Job opening in Incircular

Position: Research scientist in protein engineering

Incircular / VU University Amsterdam

Company Description

Incircular, a VU University Amsterdam spin-off, is a research-focused biotechnology startup built on a technology that enables the stabilization of proteins by chemical modification. The company is building a portfolio of stabilized proteins for various applications in therapeutics and diagnostic. Incircular is located within the laboratories of Prof. Tom Grossmann at the Faculty of Science at the VU University Amsterdam. 

Position

We are seeking an experienced molecular or chemical biologist to join Incircular. The successful candidate will work independently in an interdisciplinary team. Responsibilities will include:

  • Cloning of plasmid constructs for expression
  • Expression of proteins in bacterial, insect, yeast and human cells
  • Purification and biochemical characterization of proteins using a range of techniques, such as, affinity and size exclusion chromatography, SDS page, and mass spectrometry
  • Establishing and optimizing thermal stability assays
  • Construct engineering to optimize expression
  • Work closely with chemists, structural biologists, and business developers to advance the project
  • Create a plan of required experiments including resources and timelines to meet project milestones

Requirements

Our new team member should have the following profile:

  • PhD in a relevant area of biology or biochemistry
  • Ability to work flexibly and independently within a team
  • Analytical thinking and excellent problem-solving skills
  • Excellent organisational and time-management skills, including the ability to work to set deadlines
  • Fluency in oral and written English
  • Relevant lab experience preferably including: cloning of plasmids, protein expression and purification,development and optimization of biochemical assays
  • A strong ‘can-do’ attitude where an obstacle is a welcomed challenge.

Our offer

We offer a temporary full-time position for the duration of one year with the possibility of extension and a competitive salary commensurate with qualifications and experience. Joining our team gives you:

  • The opportunity to make a difference and play an essential role in a dynamic, innovative start-up
  • The chance to grow as a professional
  • An entrepreneurial and stimulating working environment in a growing and ambitious biotech company.
  • Applications should include a cover letter, providing a short description of their motivation, with curriculum vitae including the names and contacts of two references. Please send your application by June 20, 2021 to Saskia Neubacher, CEO (saskia@incircular.com). More information can be found on www.incircular.com and www.grossmannlab.com

Apply here: https://www.linkedin.com/jobs/view/2572601773/

Designing improved p53 peptide therapeutic using computer simulations

Dr. Atanu Maity and Asha Rani Choudhury’s joint interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Atanu and Asha are joint-first authors on the recent research paper “Effect of Stapling on the Thermodynamics of Protein-Peptide Binding”, published in J. Chem. Inf. Model. (2021). In this interview, they talk about this work and its relevance in the context of the therapeutics development.

Dr. Atanu Maity is a postdoctoral fellow at the Department of Chemistry in IIT Bombay working with Prof. Rajarshi Chakrabarti. He is from Tamluk, a small town in West Bengal. He completed B.Sc. in Chemistry from Tamralipta Mahavidyalaya in 2009. He did M.Sc. from Vidyasagar University in 2011 with a specialization in physical chemistry. After that, he joined the Division of Bioinformatics of Bose Institute, Kolkata to pursue a Ph.D. in computational biochemistry. His doctoral work was focused on the dynamics of different proteins of the Bcl-2 family involved in the intrinsic pathway of apoptosis. After his Ph.D. he moved to IIT Bombay in 2018 to join the institute postdoctoral program. In his postdoc, he is addressing different problems from physical chemistry and biological science using molecular dynamics simulation.

Asha Rani Choudhury is currently a Ph.D. student at the Department of Chemistry in IIT Bombay under the supervision of Prof. Rajarshi Chakrabarti enrolled in the year of July 2018. Prior to this, she obtained her Master degree in chemistry in 2017 securing 1st position in university level from the Department of Chemistry, Berhampur University, Odisha and Bachelor’s degree in chemistry in 2015 from Vikram deb autonomous college, Jeypore under Berhampur university, Odisha. Her research work focuses on elucidating the conformational dynamics of protein by using protein stapling procedure and its thermodynamical effect on protein-protein interaction. She is also looking forward to work in the area of free energy calculation by different enhanced sampling method and study the kinetics of protein-peptide binding. Besides her research work, she also loves running, cycling, playing chess and badminton.

Listen audio

How would you explain your paper’s key results to the non-scientific community?

Different physiological processes, taking place inside our body are complex interactions between biomolecules like protein, nucleic acid, etc at the molecular level. Any undesirable change in these interactions can lead to a situation that is known as disease. Designing medicine or therapeutic against disease is finding molecules that can interfere with this modified interaction and restore normalcy. To increase the effectiveness of these therapeutics, it is possible to design and propose modifications using computational approaches. In the present work, one such modification has been tested using computer simulations.

The interaction between two important proteins most commonly associated with cancer, mdm2 and p53, is known to serve as the guardian of the cell. Whenever there is a threat of damage to cells from stresses, different survival machinery in our body are awakened which can act upon to repair the damage. One can think of these machinery as a team of multiple players where the players are biomolecules mostly proteins. One such team is led by the protein p53 which is known for its tumor suppression activities. In response to stress, it arrests the growth of the cell and decides whether to repair the damage or to remove it. Interestingly when there is no such stress, p53 prefers to stay with its best friend mdm2, another important player in the current work.

Schemiatic

In case of the infamous disease cancer, the infected cells adopt a strategy to fool us by increasing the number of mdm2 so that most of the p53 are engaged and are unavailable for its tumor suppression function which otherwise can successfully remove/repair the infected cells.

To get rid of this, scientists have used a precise and smaller version of p53 (p53 peptide) as a therapeutic which can bind with mdm2 stronger than p53 and set the latter free for its function ensuring a healthy cell cycle. However, due to the flexible nature, the peptide lacks specificity and often fails to replace p53.

In the present work, using computer simulation we have modeled the binding of mdm2 with the p53 peptide along with p53 peptide with two modifications that reduce the flexibility of the peptide. Two specific amino acids of the p53 peptide have been replaced with hydrocarbon chains (popularly known as the stapling agent) and joined together to introduce restraint along the peptide helical axis. These rigidifications have been shown to improve the binding with mdm2 compared to wild-type p53-mdm2 binding. Detailed analyses of different binding energy components have shown significant improvement in the entropy of binding and the extent of improvement depends on the nature of the stapling agent.

A stapled peptide that can bind to RBD to mimic the binding of ACE-2 with higher affinity can be a potential candidate for therapeutic against SARS-Cov2.

What are the possible consequences of these findings for your research area?

The possible consequences of this work are two-way. The binding of the predicted stapled peptide can be studied in-vitro and in-vivo. On the other hand, the design strategy used here can be applied to find peptide therapeutics to target protein-protein interactions involved in other diseases. For example, the association of SARS-Cov2 with the host cell is initiated by the binding of Receptor Binding Domain (RBD) of SARS-Cov2 with Angiotensin-Converting Enzyme-2 (ACE-2) of human cell. A stapled peptide that can bind to RBD to mimic the binding of ACE-2 with higher affinity can be a potential candidate for therapeutic against SARS-Cov2.

What was the exciting moment (eureka moment) during your research?

Dr. Atanu Maity: The introduction of stapling agent to cross-link the side chain of amino acids of p53 reduces the conformational flexibility of the p53 peptide in an aqueous solution and its impact has been reflected in the entropy of binding. This rigidification of the peptide conformation makes us curious to ask the question, whether this can prevent denaturation? To check that, the wild type and the two stapled peptides were subjected to thermal (simulated at 330K) and chemical (simulated in the presence of 8M urea) denaturation. The wild-type peptide unfolds both at a high temperature and in the presence of urea. Interestingly, in the presence of an aliphatic stapling agent, the peptide retains its secondary structure completely at 330K and undergoes partial unfolding in the presence of urea. On the other hand, the enhanced restraints imposed by the aromatic stapling agent are able enough to endure both thermal and chemical denaturation.

Thus, the entropically favorable rigidification of the peptide by the stapling agent can successfully prevent their thermal and chemical denaturation as well. That was quite exciting!!

Asha Rani Choudhury: The p53 peptide is anchored to the mdm2 binding pocket with the help of three residues phenylalanine(F3), tryptophan(W7), and leucine(L10). Proper orientation of these residues is crucial for the binding process. A comparison of this orientation in the free peptide and the mdm2-bound peptide gives an idea about the reorganization required for the complexation process. For wild-type p53 peptide, a significant reorientation is required for complexation which is reflected in the high entropic penalty of binding. An introduction of stapling agent reduces the extent of reorientation required for complexation and the entropic penalty.

The correlation between the anchoring residue orientation in free peptide and the entropy of binding is really exciting.

What do you hope to do next?

As a continuation of the current work, we would like to use the mechanistic insights gained from this study in designing stapled peptide therapeutics to prevent SARS-Cov2 RBD and human ACE-2 binding. As our future goal, we would like to understand complex biological processes from the perspective of biomolecular dynamics. Using different tools of computational microscopy, we intend to find interesting mechanistic insights into these processes which can contribute to answer some of the unanswered questions and help to improve therapeutic strategies.

Where do you seek scientific inspiration?

Dr. Atanu Maity: The wonders of nature and the quest of understanding it, have been my constant source of inspiration. I seek inspiration from fascinating inventions and discoveries of all kinds. The survival of Deinococcus radiodurans in outer space for three years and the AlphaFold intending to solve Levinthal’s paradox amaze me equally and inspire me.

Asha Rani Choudhury: Recent commendable advances in computational chemistry and therapeutic drug designing by using different computational approaches fascinate me to challenge myself in this area and I would be very much happy if I can contribute to society in terms of designing new therapeutic strategies. Moreover, the joy of discovering new things and challenges that we come across during research keep me motivated.

How do you intend to help Indian science improve?

Dr. Atanu Maity: I think the improvement of Indian science depends on a large number of factors starting from the effort of the individual researcher to the proper allocation of resources/funds for research. As a senior researcher, I would like to focus on being part of rigorous research on interesting scientific problems and provide a proper learning platform and thorough guidance to my junior colleagues. In addition to that, I would like to be involved in improving science communication to a general audience to increase awareness about scientific research. Finally, I would put effort to improve the computational facilities at an institutional level and build more regional and national computational facilities.

Asha Rani Choudhury: Although research in India is continuously evolving, there are some elements that need to be taken care of such as computational resources, funding and motivating young researchers, etc. Furthermore,networking among different international and national institutes by organizing various conferences, webinars, workshops, and collaborations between experimental and computational laboratories will improve the overall research in India.

Reference

Maity A., Choudhury A. R. and Chakrabarti R., Effect of Stapling on the Thermodynamics of Protein-Peptide Binding. J. Chem. Inf. Model. 2021, 61, 1989−2000.

Email: atanuchem48@gmail.com, ashachoudhury95@gmail.com, rajarshi.chakrabarti@gmail.com

Lab: https://rajarshichakrabarti.wixsite.com/rajarshichakrabarti

Edited by: Anjali Mahilkar

From an offshore engineer to an AI scientist in medicine: life’s like that

Dr. Nilakash (Neel) Das is clinical data scientist, whose work lies at the intersection of artificial intelligence and respiratory medicine. As a budding researcher, he has co-authored more than 15 scientific publication. He is also an inventor of two patented algorithms for respiratory diagnostics, and is a recipient of several clinical science awards. Neel completed his bachelors from Indian Institute of Technology, Kharagpur and his masters from Technical University Delft in Netherlands. Presently, he works as a post-doctoral researcher at the laboratory of respiratory medicine and thoracic surgery at KU Leuven, Belgium. He also collaborates extensively with ArtiQ, a spin-off company of their laboratory, in translating AI research into clinical practice.

Listen audio

As I reflect on my PhD journey, I feel that it was the best decision I have ever made in my life.

In another lifetime, almost six years ago, I was designing oil platforms and vessels. Little did I imagine then that my life was about change its course. I could feel the drift, my conscience plunging into an internal turmoil that the work I did was irreconcilable with the global havoc wrecked by climate change. Therefore, I just jumped ship after spending almost a decade in that field.

Around the same time, the real potential of artificial intelligence (AI) was starting to unravel. Rapid developments in deep learning, which had burst into public consciousness such as Alexnet(Krizhevsky et al. 2013), signalled the dawn of a new era. Algorithms were now capable of automating tasks that otherwise require the quintessential human perception. In-fact, I even tried some of my half-baked AI ideas in master’s thesis not to the amusement of my supervisors who were classical physicists.

Masters graduation in offshoroe engineering (2016)

I knew, in my heart of hearts, that a doctoral apprenticeship in applied AI in medicine, was the perfect opportunity to learn and to make a meaningful contribution to improve human lives. It would have allowed me to take a deep dive into the exciting world of data science. These are good thoughts to have. But why would a field as conservative as medicine would accept me when I have ‘oil’ on my hands?

In the summer of 2016, my co-promotor had advertised a position that he was looking for a PhD candidate to build mathematical models on spirometry (a medical test to monitor respiratory health). I had no idea what spirometry was, but that did not stop me. That was the only position I had applied that summer, and I was overjoyed when I received a call for an interview.

I clearly knew that there were candidates who were far more qualified. To ‘sell’ my engineering skills, I came prepared with a few slides on how to fit a non-linear model on observed lung pressure-volume loops (although I had no idea then, on the physiological mechanism!). I vividly remember that joyful moment when I received an email congratulating me! It had come after a long time of stressing about my future, and my dwindling finances. Years later, my co-promotor confided to me that he did not consider my CV initially as it was so alien to the respiratory field, only to have been convinced by a friend of his to take a second look as I had some mathematical skills. I thank that person whole-heartedly!

However, the PhD journey was never a smooth ride. In the beginning, the learning curve was overwhelming. Not only I had to educate myself on classical statistics and AI, I also had to soak in an enormous body of literature on lung physiology and respiratory medicine. I am indebted to all the amazing online resources and MOOCs that made my ride much smoother. Without these resources, I shudder to think if I could have even completed my PhD.

Nonetheless, real challenges arose when the time came to communicate my results. Initially, my presentations were highly technical/mathematical which would amuse or even frustrate my colleagues, who were mostly clinicians and biologists. It was the same story with my research manuscripts that were being rejected by one journal after another. It was at these lowest moments that my supervisors played a big role in shaping my attitude. They taught me how to transcend from thinking only about the technical facets of an algorithm, to deeply caring about the clinical aspects as well. I also attended countless presentations to understand the nuances of scientific communication. This led to a dramatic transformation in my approaches, resulting in over 15 peer-reviewed articles (including Nature), 2 patents, 3 clinical science awards and several public presentations.

PhD graduation in AI in respiratory medicine (2021)

As I reflect on my PhD journey, I feel that it was the best decision I have ever made in my life. What started out as an avenue to learn more about AI, became much more. I learnt to do science. Asking the right questions, translating a gut feeling into a hypothesis, using data to prove or disprove it, has been an exercise of enormous intellectual stimulation. It imbibed in me the scientific rigors of analysing, interpreting, arguing and communicating clearly and logically. The most important of all, it taught me to be humble about the fact that I know only an infinitesimal amount within the infinitely large corpus of knowledge. An opportunity, such as a doctoral apprenticeship, in which my contribution led to a minuscule gain in the understanding of human health, gives me immense pleasure.

Edited by: Nivedita Kamath

Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters

Dr. Shabareesh Pidathala’s interview with Bio Patrika hosting “Vigyaan Patrika”, a series of author interviews. Dr. Pidathala received his schooling in a small town called Bellampaly in the present state of Telangana. As a school kid, he had always been interested in chemistry and biology. This interest directed him to do a bachelor’s in botany, chemistry and biotechnology from Sri Venkateshwara University, Tirupati, Andhra Pradesh. He went on to pursue a master’s in biotechnology from Pondicherry central university. He continued his scientific quest as a Ph.D. scholar at the National Institute of Immunology, New Delhi, where he worked at the interface of peptide chemistry and protein biochemistry. He designed, synthesized and carried out structure-activity studies on (glyco)peptides that can inhibit human thrombin, which has the potential to prevent undesirable blood clotting. Post Ph.D., he joined Dr. Aravind Penmatsa’s lab at the Indian Institute of Science to understand the structural basis of proteins involved in neurotransmitter transport. Here, Shabareesh talks about his work on Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters published in Nature Communications.

Listen audio

How would you explain your paper’s key results to the non-scientific community?

Communication between nerve cells (neurons) happens with the help of chemicals called neurotransmitters. These chemicals are released from one nerve cell and bind to the proteins present on adjacent nerve cells’ surfaces to further relay the information. Excess neurotransmitters released are transported back into the nerve cell. For this uptake of the neurotransmitter, dedicated transporter proteins are present in the cell membranes of nerve cells; they are called neurotransmitter transporters. These transporters play a crucial role in regulating the strength and duration of communication between neurons. Noradrenaline (also known as norepinephrine) is a neurotransmitter that is important in regulating alertness, pain sensation and arousal. Increasing the levels of noradrenaline in the spinal cord was found to relieve chronic pain. One way of doing this is by blocking the neurotransmitter transporter from uptaking the released noradrenaline. Several prescribed medications to treat chronic pain conditions act by blocking the transport of noradrenaline by neurotransmitter transporter. These medications include S-duloxetine, milnacipran and tramadol, which are commercially available as Cymbalta, Savella and Ultracet that are used to treat fibromyalgia, neuropathic pain and post-operative pain, respectively. Our study shows at an atomic level how a neurotransmitter transporter recognizes noradrenaline and how duloxetine, milnacipran, and tramadol block the transporter. Through these structural studies we identified a region in the binding pocket of the transporter crucial for the recognition of noradrenaline and the blockers. These results can help in designing effective pain medications with reduced side effects.

Schematic

Our study provides a detailed structural understanding of noradrenaline recognition by a neurotransmitter transporter.

What are the possible consequences of these findings for your research area?

In the neurotransmitter transporters research field, structures of only three major neurotransmitter transporters are known so far, they include dopamine transporter, serotonin transporter and glycine transporter. Noradrenaline and dopamine are chemically very similar and yet have very different physiological effects. So far, it has not been understood structurally how a neurotransmitter transporter recognizes noradrenaline and how it is different or similar to that of dopamine recognition. Our study provides a detailed structural understanding of noradrenaline recognition by a neurotransmitter transporter. Besides this, it also provides structural insights into recognizing clinically significant blockers of noradrenaline transport used to manage chronic pain conditions. Our structural observations resulted in the identification of a novel region in the transporter, which is crucial for the specific recognition of noradrenaline and the blockers. This information can be essential in designing effective medications to treat chronic pain.

What was the exciting moment (eureka moment) during your research?

Navigating through this project was an exciting journey, and there were several eureka moments for me. We determined six different structures in this project; seeing the electron density map for every structure had been equally exciting. If I have to name one, then it would be observing the clear electron density map for noradrenaline. Working with noradrenaline is tricky as it gets oxidized easily, leading to the formation of undesirable quinones. Hence, crystallization experiments were done with minimal illumination at 4°C along with an anti-oxidant to prevent the oxidation of noradrenaline. Seeing an unambiguous electron density for noradrenaline was very gratifying and gave us the much-needed confidence to execute this project successfully.

What do you hope to do next?

My journey in the field of structural biology continues. Understanding how biomolecules work at an atomic scale always excites me. Electron cryo-microscopy (Cryo-EM) is dominating the field of structural biology and is making it possible to study unimaginably large molecular complexes both in vitro and in situ. I am very keen on pursuing my future research in this field.

Where do you seek scientific inspiration?

My interest in science started during my childhood. My father used to talk to me about Dr. A.P.J. Abdul Kalam’s painstaking journey in his pursuit of scientific knowledge. It was the same time when I used to save my pocket money to purchase a monthly edition of “Science Reporter,” a scientific magazine being published by CSIR. It used to provide a gist of the latest research done in premier research institutes around the globe. These initial seeds of interest directed me to make scientific research my career option. Whenever I hit a roadblock in doing my research, one thing that always motivates me is this quote by Madam Curie “Nothing in life is to be feared, it is only to be understood.” I believe scientific research is the only way to understand this world better and fear less.

How do you intend to help Indian science improve?

I believe the Indian scientific diaspora is very diverse and robust. Given a conducive environment to foster rich collaborations, we can easily be the global leaders in scientific research. My contribution to Indian science will continue; hopefully as an academician, and I will strive to initiate and be part of any appropriate collaborative research. Moreover, it’s our collective responsibility to communicate science better and cultivate the habit of scientific inquiry among people.

Reference

Pidathala, S., Mallela, A.K., Joseph, D. et al. Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters. Nat Commun 12, 2199 (2021). https://doi.org/10.1038/s41467-021-22385-9https://www.iisc.ac.in/how-does-chronic-pain-medication-block-noradrenaline-transport/

Highlight: https://www.iisc.ac.in/how-does-chronic-pain-medication-block-noradrenaline-transport/

Penmatsa lab: https://aplabmbu.weebly.com/

Edited by: Vikramsingh Gujar

Role of centromeres in evolution and karyotype diversity in Candida auris

Dr. Aswathy Narayanan’s interview with Bio Patrika hosting “Vigyaan Patrika”, a series of author interviews. Dr. Narayanan pursued her doctoral research in yeast genetics, under the guidance of Dr. Md. Anaul Kabir in National Institute of Technology Calicut, Kerala. Her doctoral research was focused on the eukaryotic protein folding machinery – its functions and interactome that are conserved across species. She joined Prof. Kaustuv Sanyal’s group in Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) as a postdoctoral researcher in 2016. Incidentally, around the same time, Candida auris emerged as a fungal pathogen of concern worldwide. Under Prof. Sanyal’s mentorship, she shifted her model organism from the docile Saccharomyces cerevisiae to the multidrug-resistant superbug C. auris. She is interested in multidrug resistance, stress response pathways, and karyotype evolution in fungal pathogens and aspires to identify pathogen-specific pathways/genes which can be translated to clinical practice in the future. She currently works as Scientist-B in a multicentre project funded by Indian Council of Medical Research involving JNCASR, Post Graduate Institute of Medical Education & Research (PGIMER) and Amity University Haryana. Outside the lab, she divides her time between music, books, and her three dogs. Here, Aswathy talks about her work on Functional and comparative analysis of centromeres reveals clade-Specific genome rearrangements in Candida auris and a chromosome number change in related species published in mBio.

Listen audio

How would you explain your paper’s key results to the non-scientific community?

Fungal pathogens cause infections in immunocompromised patients especially in intensive care units of hospitals. In 2009, Candida auris, a novel fungal pathogen was isolated in Japan. Within a decade, it was isolated from patients in hospitals in different parts of the world, and it is difficult to identify, treat and eradicate, as it is resistant to major antifungals used in clinics. Interestingly, C. auris isolated in different parts of the world exhibit different properties – they are grouped as clades based on their geographical isolation.

Fungal pathogens can alter their genomic content that helps them to adapt to different environmental and host niches. Previous studies from our lab have shown that most of these events include centromeres – the regions on the chromosome that facilitate the distribution of the genome content equally to the daughter cells during cell division. There are previous reports of the C. auris genome being dynamic. What are the properties of centromeres in C. auris? Are they different in the geographical clades and do they play any role in this diversity observed within a species? These are a few questions that intrigued us.

The first step was to identify the location of centromeres on the chromosomes. We tagged a histone protein that localizes specifically to the centromeres. Tracing its localization using chromatin immunoprecipitation and microscopy helped us to locate the centromere positions. We did these experiments in all geographical clades- and the centromeres had the same properties across clades. But, at the chromosome level, the analyses revealed interesting results. The East Asian clade, consisting of isolates mainly from Japan and Korea, had all chromosomes and centromere locations shuffled (1). This clade is the “odd-one-out”; they are sensitive to antifungals and are incapable of causing bloodstream infections.

Figure 1: Candida auris emerged as different geographical clades, out of which the East Asian clade (red) consists of atypical isolates that are drug-susceptible and incapable of causing invasive infections.

We then expanded the panel of our study and looked at a species complex closely related to C. auris, called Candida haemulonii complex. We identified centromeres in these species and found they had similar centromere properties, and gene neighborhoods. Though different species, they shared several patterns with C. auris – a centromere inactivation leading to a chromosome number change, chromosome breaks near centromeres – all hinted at the existence of a common ancestor shared by C. auris and C. haemulonii complex. Different species diverged from this ancestor and clade 2 diverged further, making it different from other clades.

Figure 2: Chromosome number variation in closely related species is mediated by a centromere inactivation event. Two centromeres placed on the same chromosome results in inactivation of one of the centromeres in a group of related species (highlighted in gray).

What are the possible consequences of these findings for your research area?

One of the observations of this study is that centromeres are genomic loci that evolve fast. So far, we knew this to be the case between species like C. albicans and C. dubliniensis (2). Now we know that the same is true within a species. This expands the notion of centromeres being just the primary constrictions involved in cell division. We uncover an instance of centromeres mediating chromosome number changes that is a major event in the emergence of a new species. It is tempting to consider centromeres as active participants in speciation.

We uncover an instance of centromeres mediating chromosome number changes that is a major event in the emergence of a new species.

What was the exciting moment (eureka moment) during your research?

The exciting moment was when we realized that two centromeres which were present on two different chromosomes in other related species were present on one chromosome in C. auris. This would imply that one of them must have been inactivated, as a chromosome can have only one functional centromere to be stable. We also know that sexual reproduction is responsible for the diversity we see in the living world. It was fascinating to think that nature has other raw materials for generating diversity in these asexual fungi and leaves some footprints of these events behind; we stumble upon them millions of years later.

What do you hope to do next?

Fungi are known for the chronic and persistent infections they cause. The fact that a fungal species emerged and spread across the whole globe within a short span is alarming- I would like to understand the factors that made this possible. We are familiar with fungal species like Candida albicans that grow at high temperatures; fortunately, we are armed with antifungals to combat these infections. We also know that some fungal species exist that are generally resistant to these antifungals, but the incidence of those infections is low. But C. auris is a species that has both the features- it can grow at high temperatures and exhibits resistance to antifungals which makes it a potential superbug. I plan to study the molecular mechanisms underlying these features.

Where do you seek scientific inspiration?

The fact that life originates and perfects itself under diverse conditions never fails to amuse me. It happens from deep waters to hot springs. Every living creature operates like a tiny unique machine – internally programmed and running for a time after which wear and tear sets in. During this process, it faces several adverse conditions and adapts persistently to stay alive. The intricacies of this regulation are a source of constant inspiration. Apart from this, Barbara McClintock, the scientist who was way ahead of her time, remains an inspiration.

How do you intend to help Indian science improve?

One aspect that the study on C. auris made me realize is that there are several endemic angles to a research question. We know that the Indian isolates of C. auris have properties of their own. I feel that microbiological research, especially when dealing with pathogens, can include these angles. This will help us to directly translate our understanding of molecular mechanisms to clinics- either as drug targets or diagnostics. I aim and hope to contribute to this aspect in the future.

References

1.        Narayanan A, Vadnala N, Ganguly P, Selvakumar P, Rudramurthy SM. 2021. Functional and comparative analysis of centromeres reveals clade-Specific genome rearrangements in Candida auris and a chromosome number change in related species. mBio 12:1–23.

2.        Padmanabhan S, Thakur J, Siddharthan R, Sanyal K. 2008. Rapid evolution of Cse4p-rich centromeric DNA sequences in closely related pathogenic yeasts, Candida albicans and Candida dubliniensis. Proc Natl Acad Sci U S A 105:19797–19802

Edited by: Anjali Mahilkar

Learn more about Prof. Kaustuv Sanyal’s research group here:

https://molecularmycologylab.wixsite.com/kaustuv

Conformational flexibility and structural variability of SARS-CoV2 S protein

Miss Ishika Pramanick’s interview with Bio Patrika hosting “Vigyaan Patrika”, a series of author interviews. Ishika is from Kolkata, West Bengal. She completed her schooling at Ramakrishna Sarada Mission Sister Nivedita Girls’ School, Kolkata. She pursued her B.Sc degree in Microbiology from Scottish Church College, Kolkata and M.Sc degree in Microbiology from the University of Calcutta. Currently, Ishika is a graduate student in the Molecular Biophysics Unit, Indian Institute of Science, Bangalore. She is working on the single-particle cryo-Electron Microscopy (Cryo-EM) field under the supervision of Dr. Somnath Dutta. Here, Ishika talks about her recent work on Conformational flexibility and structural variability of SARS-CoV2 S protein published in Structure.

Listen audio

How would you explain your paper’s key results to the non-scientific community?

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV2) is one of the deadliest viruses responsible for one of the worst global pandemic in recent times. Spike glycoprotein (S-protein) resides on the surface of SARS-CoV2. It interacts with human angiotensin-converting 2 (hACE2) receptor protein through its Receptor Binding Domain (RBD) to help the virus infect the human body. Spike glycoprotein structure is highly susceptible to pH      change; therefore we investigated whether there is any conformational flexibility in S-protein with changes in pH. We selected pH 7.4 (physiological pH), and pH 6.5 and pH 8.0 (near physiological pH) for the study. S-protein is mostly present in two states, (i) “open state” and (ii) “closed state”. The transition from a closed state to an open state regulated by RBD movement, which is important for the proper binding of S-protein to hACE2 receptor. In other words, the higher the number of open state confirmation of S-protein exists, the more relevant for viral infection. We show a significantly higher proportion of open state conformation (68%) at pH 7.4 than at any other pH, which explains why the virus successfully infects humans at physiological pH. We have resolved open state, closed state of S-protein at and near physiological pH. Not only open and closed state, we have also resolved the intermediate conformations of S-protein which illustrates the flexible nature of RBD and NTD. We also provide evidence for heterogeneity in both open and closed conformations, meaning not all S-protein orient in the same way. This heterogeneity generates different binding sites for the neutralizing antibodies to neutralize the virus. Overall, we demonstrate the inherent structural flexibility of S-protein at different pH. 

Schematic

Our findings have the potential to aid the future development of therapeutic agents against the virus considering the versatility of the S-protein structure.

What are the possible consequences of these findings for your research area?

This is the first time, we observed various intermediate conformations of S-protein at near physiological pH, and a high proportion of open state of S-protein at physiological pH using cryo-EM. Our hypothesis is, this pH dependent alteration in S-protein structure could be the mechanism with which the virus evades the immune response. Our results might assist us to understand why there are so many candidates for neutralizing antibodies to treat the infection and why the vaccines available in the market today that target S-protein doesn’t have an efficacy of 100%. Our findings have the potential to aid the future development of therapeutic agents against the virus considering the versatility of the S-protein structure.

What was the exciting moment (eureka moment) during your research?

It’s difficult to pinpoint any particular instance as the eureka moment as I consider the whole experience of this research as a eureka moment. From ideation of the project to taking stunning cryo-EM images to spending many sleepless nights writing up the manuscript was a great learning phase for me. Also, the experience of data processing with smart and wonderful people has been a fantastic journey. We are determined to continue on this journey to unravel more aspects of this deadly virus to help humanity.

What do you hope to do next?

Next, we are in the process of setting up collaborations to focus on deciphering the interaction of S-protein with different drug molecules, neutralizing antibodies, and synthesized peptides at physiological pH. Outcomes from these investigations could pave the path for the inception of new therapeutics against the fatal SARS-CoV2 virus.

Where do you seek scientific inspiration?

To quote Dr. A.P.J. Abdul Kalam, “Dream is not that which you see while sleeping, it is something that does not let you sleep.” The excitement of learning new things is what motivates me to get out of bed every morning. I seek scientific inspiration from all the women working in STEM (Science, technology, engineering, and mathematics), as they work hard to make the world a better space.

How do you intend to help Indian science improve?

Right now, it is too early for me in my career to answer this question. However, with my work and determination, I am hoping to inspire the future generation, particularly women, to take STEM as a career and work towards the welfare of society.

Reference

Ishika Pramanick, Nayanika Sengupta, Suman Mishra, Suman Pandey, Nidhi Girish, Alakta Das, Somnath Dutta. Conformational flexibility and structural variability of SARS-CoV2 S protein. Structure. 2021. PMID: 33932324, PMCID: PMC8086150, DOI: 10.1016/j.str.2021.04.006

Edited by: Sukanya Madhwal