Bio Patrika interviews Dr. Sharma on his thoughts about “CRISPR/CAS9 and regulation of flavonoid biosynthesis in plants”

Mr. Ashish Sharma’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Mr. Sharma is currently working as CSIR-Senior Research Fellow at CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh. He has completed his PhD under the supervision of Dr. Prabodh Kumar Trivedi, the Director, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow and is waiting to defend his thesis. Mr. Sharma published paper titled “Primary transcript of miR858 encodes regulatory peptide and controls flavonoid biosynthesis and development in Arabidopsis” as the first author in Nature Plants journal (2020).

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

Our research published in Nature Plants reports the identification and functional characterization of microRNA encoding peptide (miPEP858a) in model plant Arabidopsis thaliana. Besides, this study also demonstrated the possibility of using CRISPR/Cas9 as an innovative tool for the functional analysis of single members of miRNA families in plants. These peptides have significant potential to control flavonoid biosynthesis. Flavonoids are a class of ubiquitous plant secondary metabolites with numerous health-promoting properties including anti-cancer, anti-inflammatory and anti-viral activities. These are the vital components of the human diet.

“[…] CRISPR/Cas9 as an innovative tool for the functional analysis of single members of miRNA families in plants.”

Nobel Prize 2020 in Chemistry for the Discovery of ‘Genetic Scissors’ called CRISPR/Cas9 awarded to Emmanuelle Charpentier and Jennifer A. Doudna.

To explore the detailed role of these peptides in plants, we generated mutant plants defective in miPEP858a along with mutants of both the members of miR858 family through the CRISPR/Cas9 approach. Interestingly, hindering miR858a activity by editing miPEP858a resulted in the up-regulation of genes involved in flavonoid (flavonols and anthocyanins) biosynthesis at the cost of lignin production.

We also developed transgenic lines overexpressing miPEP858a, which elucidated that this peptide also promotes plant growth and development in association with auxin. We performed complementation studies using synthetic miPEP858a peptide to gain a more in-depth insight into its function. Exogenous application of this peptide to miPEP mutant plants was able to complement the lost function and rescue the growth phenotypes of the peptide, confirming its vital role in regulating miR858a expression. This study adds a new layer of regulation of miRNA and confirms the role of miPEPs in plant growth and development.

Biogenesis of miPEP. Schematic representation of biogenesis of miPEP, miRNA gene transcribed by RNA pol II to form pri-miRNA, which is subsequently processed to form the mature miRNA. A part of pri-miRNA which have functional ORF, encodes small peptide known as microRNA-encoded peptide (miPEP), which increases the transcription of miRNA gene.

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

Till recently, the biological roles of miRNAs were explained either by over-expressing the miRNA under the control of a strong promoter like CaMV35S or through down-regulating/sequester the miRNA with RNAi/target mimicry approach and studying the phenotypic and molecular analysis of these transgenic plants.

As miPEPs are anticipated to be functional only in cells where the specific encoding miRNA is expressed, this would avoid non-specific phenotypes associated with ectopic expression of miRNAs, which is one of the major disadvantages of the approaches mentioned above. Thus, the application of synthetic miPEPs or their transgenic overexpression will help in functional analysis of individual members of the miRNA families.

The findings of our study also suggest that the exogenous application of miPEPs can evade the tedious and complicated procedures involved in the production of transgenic plants and is also easy to implement. The possibility to use miPEPs in exogenous treatments owing to their specificity provides a new and powerful tool to study the role of miRNAs and can significantly modulate key plant developmental processes with high agronomical important crops like rice, wheat, maize and tomato etc. These findings provide an additional layer of miRNA regulation.

“[…] the exogenous application of miPEPs can evade the tedious and complicated procedures involved in the production of transgenic plants and is also easy to implement.”

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

Establishment of CRISPR/Cas9 in the lab by mutating AtPDS gene (a carotenoid pathway gene), which upon successful editing displayed striking albino phenotype in leaf and over-all plant, that for me was the most exciting moment. Later, the miR858 family and miPEP858a were mutated, and these edited plants displayed remarkable pinkish/purple colour due to enhanced accumulation of anthocyanin (which has medicinal properties) was the most exciting moment. 

What do you hope to do next?

This study elucidated the in-depth role of non-coding RNAs demonstrating that they are capable of producing regulatory peptides that possess significant biological functions, mostly associated with agronomical traits like plant height, seed size, plant growth and development and synthesis of secondary metabolites (flavonoids and anthocyanin).

Our next big aim is to elucidate the in-depth regulatory mechanisms of miPEPs and its cross-talk with other regulatory molecules to gain further insight into how this small peptide regulates miRNAs as well as the crucial biological processes in plants. We are also looking forward to the identification of newer miPEPs and other regulatory small peptides with high agronomical values. 

Where do you seek scientific inspiration?

After the successful editing of PDS for the establishment of CRISPR/Cas9, I felt that I am moving in the right direction, and later, we edited different members of the miR858 family in Arabidopsis thaliana. During that time, research on discoveries of small plant peptides and the mighty roles they play was the new interest to scientists worldwide. These few amino-acids accounts for a drastic change in plant physiology and adaptation motivated me to look for miRNA encoded peptide for miR858. I was incredibly fortunate to identify a functional miPEP and then carried out its detailed analysis. Throughout my research, I was lucky enough to be continuously inspired by my supervisor, Dr. Prabodh Kumar Trivedi, who always taught me that to conduct adequate research; we ever need to have a good imagination, persistence and patience.

How do you intend to help Indian science improve?

This peptide signifies an additional and excellent example of peptides derived from previously known as non-coding RNA with powerful biological functions whose encoding sequences were previously hidden in the genome. The identification of miPEPs unlocks new means of studying the corresponding miRNA families and therefore, would help in improving desired traits and yields in agronomical crops.

Also, exogenous application of synthetic miPEPs stimulates the expression of the corresponding miRNA owing to its specificity. The miPEPs, in this regard, can be used as an alternative tool to enhance the crucial agronomical traits of crops. However, the application of synthetic peptides in fields would be costly but beneficial for plants, which are way difficult to be manipulated and thus avoiding the need for genetic modifications/ cloning and other complex processes.

Applications and potential of miPEPS in crop improvement. miPEPs could be effectively used in crops to improve traits, such as yield, plant architecture and stress tolerance.

Reference

Sharma A, Badola PK, Bhatia C, Sharma D, Trivedi PK. Primary transcript of miR858 encodes regulatory peptide and controls flavonoid biosynthesis and development in Arabidopsis. Nature Plants. 2020. doi: 10.1038/s41477-020-00769-x.

Ashish Sharma email: sharmaashish23march@gmail.com

Fibroheal offers innovative silk-based biomaterial for wound care

Fibroheal Woundcare Pvt. Ltd founded by Vivek Mishra (Founder, Director & CEO), Bharat Tandon (Co-founder and Director) and Subramanian Shivaraman (Co-founder & Director) in 2017. Based in Bangalore, Fibroheal is a biotechnology oriented healthcare startup with focus on the use of biocompatible silk proteins based biomaterial for advance & active wound care management.

What was the vision and inspiration behind setting up Fibroheal? How did you come upon the technology?

Silk is one of the oldest fibers known to man, it has a history as the richest fabric itself! There are silk sarees which are more than 100 years old and are used for many generations even today; which highlight its excellent fabric and material property. When it comes to exploring its non-textile use, the application of silk in the medical field started in the 19th century when it replaced traditional metal wires as a suture material. 

Today, silk is studied extensively for its biomedical use. It is the most diverse, model biomaterial due to its remarkable properties like high mechanical strength, biocompatible, non-cytotoxic, thermo-stable, low-immunogenic and biodegradable.

India is the second-largest silk-producing country in the world. Karnataka alone produces nearly 70% of the country’s total mulberry silk. Sericulture farming provides jobs for about 10.6 Lakh people in Karnataka directly and indirectly. More than 1.2 Lakh families in Karnataka are depending on sericulture and mulberry cultivation. But a large amount of silk is wasted in the value chain from reeling to weaving which can be utilized to develop products for biomedical use. More than 80 lakh families are dependent directly and indirectly on silk. 

We at FIBROHEAL WOUNDCARE PVT LTD always wanted to bring socio-economic multiplier effect with our innovation and work i.e. from Silk growers (farmers side), surgeons (user side) and patients. Since our facility is situated very close to Silk city of Karnataka, having a largest market of silk cocoons in Asia, the sourcing of good quality silk/raw materials for our manufacturing is like at our doorstep & ease. We work on the non-textile application of silk, utilizing the waste and by-products of silk production to develop innovative, low-cost products for patients. At the same time, we are creating employment for many, enhancing farmers’ income and improving the economics of silk production in India.

Secondly, due to increasing ageing population worldwide and the high prevalence of lifestyle diseases and chronic diseases among the elderly and high cost of treatment, we wanted to work on newer biomaterial which can be cost-effective and affordable for patients and at the same time ease the work of doctors/nursing staff in the hospital.

Vivek Mishra, Bharat Tandon and Subramanian Shivaraman (Left to right).

Additionally, we have on-board, two eminent stalwarts to guide us as co-founders, mentors and advisors. 

Mr. Bharat Tandon – Founder of Vetcare (Animal nutrition and health company) which was acquired by Provimi and later by Cargill.

Mr. Subramanian Shivaraman – Co-founder of the second-largest sutures company in India called Sutures India (having many sutures along with silk sutures) now known as Healthium Medtech.

Thirdly, the need gap which exists in the present market scenario, more than 85% of MD are imported in India. Being the second-largest producer of Silk in the world, second most populated in the world and depending solely on imports to cater to various wound healing requirements initiated the thoughts. Silk being the most versatile, perfectly biocompatible material and its abundant availability in Karnataka made it an ideal candidate for our research. Moreover, a lot of research happened at various institution levels on silk as its biomaterial applications. We saw the economic multiplier potential of silk – farmers, hospitals and patients. All this put together guided us our path to develop a product which can have a substantial economic impact, can bring down imports significantly and product performance is much superior to the existing imported and marketed products while being cost-effective.

“[…] we are creating employment for many, enhancing farmers’ income and improving the economics of silk production in India.”

What were the challenges faced in the initial years? Any competition?

Like any other startup, we also experienced several challenges as mentioned below.

1. Funding: Initially, we have started our operations in 2017 with internal investment. At later stages, we have been successful in raising some small angel investment, government grants and started generating some revenues from sales.

2. Right hiring: Attracting good talent was very difficult as a startup company due to many limitations. Later we started hiring by good references, going back to colleges, using our connects and old colleagues helped us with the right hiring. And right now we have a robust and dynamic team which is our company’s core strength.

Fibroheal Woundcare Team

3. Regulatory clarity: To address this we have identified few professionals who have guided and helped us in getting the regulatory clearance, which was one of our prime most requirements.

4. Maintaining liquidity in the company: We have a committed and experienced sales team having excellent hospital and distribution network to operate at ground level where we started selling our products and generating revenues by getting regular business.

5. Research & Technical guidance: We have collaborated with reputed institutions like IISc Bangalore, IIT Guwahati for technical support and guidance for research and development and Dept. HOD`s of few reputed institutions as medical advisors, mentors and key opinion leaders to guide us on market need, products and its performance. 

Our major competitors are 3M, Smith & Nephew, ConvaTec, Coloplast, KCI, J&J, Mölnlycke Health Care and Hollister. 

We have registered under Startup India, Startup Karnataka, and this helped us in getting benefits in various Govt. tenders to get an exemption for a turnover clause for participation. Since we are registered under Startup India, we listed our company and products on GeM (Govt. e-market place). Additionally, Dept. of Biotech, Govt. of India through BIRAC has supported us in a lot of initiatives. We were also winners of ELEVATE 100, 2019 organised by Startup Karnataka under Dept. of IT, BT & ST, Govt. of Karnataka as one of the most innovative biotech startups and received a R&D grant to support our research. CCAMP under a BIRAC program has supported us through LEAP (Launching Entrepreneurs for Affordable Products) scheme. 

What are its applications? 

We offer a wide range of silk protein derived wound dressings and products to treat different wound types and conditions. 

Value propositions to the customers:

  • Indigenously developed a solution for wound healing (Natural biomaterial, bio-compatible and safe).
  • Aids faster healing & wound closure.
  • Significant reduction in the cost of treatment for patients.
  • Offers scar reduction while participating in active healing.
  • Faster turn-around rates and decreased load on hospitals.
  • Improves the quality of life of people post healing.
  • Affordable and easily accessible.

Describe your product line and production capacity? Any eco certifications?

We are one among the few companies working across the globe on this versatile biomaterial called silk proteins. We have indigenously developed patented technological products in our basket. Our research work is well appreciated by the government and supported by Grants and Awards. We offer affordable and cost-effective solutions for wound management. We have successfully launched nine products in the market, and our products are used by renowned surgeons and doctors in more than top 50 major reputed hospitals (like Government, Private, Medical College, Defense, Railway and Autonomous etc.) across India, to treat the most complicated wounds and are happy with the results.

We aspire to be one of the most comprehensive wound care Indian company catering to the entire wound care continuum. We offer products for active and advanced wound care management. Our Fibroheal range of products are used to treat all types of wounds such as clean wounds, non-infected wounds, infected wounds, challenging to heal and slow or non-healing wounds etc. Our products come in different forms such as film, sheet, mesh, foam, powder, ointment, gel etc. 

We have built a state-of-art facility, which is ISO 13485 compliant and got a CDSCO license for manufacturing. We have installed automated machines to increase the production capacity with a 10x jump in output (approx. 1 Lakh units per month).

We are an environmentally friendly enterprise, manufacturing the completely biodegradable product. The entire operations are managed in such a way so that there is no minimal impact on the local or global environment.

Your insights on the sustainable healthcare product market in India? Has Covid 19 changed the scenario?

Both globally and in India, the Wound care market is growing at a significant pace year after year with increasing population, lifestyle diseases (diabetes), disposable incomes etc. Moreover, in the current market scenario, there is an emerging need gap, with most medical devices being imported into India, resulting in high import costs. We see a huge potential and sustainable business for products which are indigenously developed, cost-effective and fulfilling the needs of the wound care market.

Post COVID, our business is also impacted, and we hope to bounce back once the situation improves. We have received excellent responses from the medical fraternity, and they are optimistic and positive to support research-driven Indian startups who are making meaningful products and solving genuine problems. However, even during the pandemic, lock-down phase and when business conditions are challenging, we stood by our people, supported them by paying them fully without any pay-cuts or lay-offs. We hope to make a lot of difference and aspire to be the most comprehensive wound management company in the coming time.

Do you export your products? If yes, to which countries?

As of now, we are operating in the domestic market. However, we are in the process of getting CE approval to enter the export market by 2021.

What are your future plans?

We are on a growth trajectory, and our products are well accepted in the Indian market, users are satisfied with the performance of the products, and received fantastic response from surgeons as well as patients. Few newer concept-based products are in the pipeline which we are aiming to launch by 2021. Additionally, we are in the process of getting CE approval for our product range. Post CE certification, we are planning to expand our business & hit the international market, including developing and developed countries. 

Address: Fibroheal Woundcare Pvt. Ltd., IS-21 KHB Industrial area, Yelahanka New Town, Bangalore, Karnataka 560064, India. Email: fibroheal@gmail.com. Website: http://www.fibroheal.com/.

Job opening in Meticulous Research

Meticulous Research is hiring for a Research Associate – Healthcare position.

Qualification: MBA, B.Pharma, M.Pharma

Skills required: Good communication

Years of experience: 0-1 year

Location: Wakad, Pune

Immediate joiner will be preferred.

If you are interested, please share your updated resume to bhagyashree.t@meticulousresearch.com.

Job opening in Affigenix – Chennai

Affigenix Biosolutions Private Limited is hiring.

Qualification: Undergraduate and Master’s degree from Biochemistry, Microbiology and Biotechnology field.

Responsibility: Development of novel diagnostic kits.

Location: Diagnostic kit manufacturing facility located at Alathur near Chennai, Tamil Nadu.

Check Affigenix website for more information: www.affigenix.com.

If interested, please share your updated CV with cover letter to info@affigenix.com.

Bio Patrika interviews Dr. Sengupta on her thoughts about “directionality of proteolysis”

Dr. Bhaswati Sengupta’s interview with Bio Patrika hosting “Vigyan Patrika“, a series of author interviews. Dr. Sengupta is currently working on Nucleosome dynamics and chromatin remodelling using smFRET as a postdoc in the Department of Chemistry at Pennsylvania State University, USA. She did her PhD from the lab of Prof. Pratik Sen, Department of Chemistry, Indian Institute of Technology Kanpur, India. She published last part of her PhD work titled “Fluorescence correlation spectroscopy as a tool to investigate the directionality of proteolysis” as the first author in International Journal of Biological Macromolecules (2020).

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

Proteins are a long chain of amino acid molecules connected by a peptide bond. Proteolysis is a significant part of the digestion system where the protein gets cleaved into smaller peptides. In this paper, we have used human serum albumin (HSA) as a model protein and papain, chymotrypsin, and trypsin as enzymes to digest HSA. Being a long-chain protein, HSA can be cleaved from either side. In this work, we have used fluorescence correlation spectroscopy (FCS) to understand exactly from which side these enzymes cleave HSA. FCS enables us to measure the change in the size of the fluorescently labelled fragments. As the digestion proceeds, the size becomes smaller, which we measure by FCS. By preferentially labelling to two sides of the protein chain with fluorescent dyes, we can see from which side the digestion process starts. The principle of the measurement is shown in Scheme 1.

Scheme 1: Monitoring protein digestion by single molecule technique.

HSA is a large protein having three domains. For HSA, we found out that digestion starts from the domain I for all three enzymes under consideration. Fluorophore 1 (TMR) slows the faster decrease of size as compared to fluorophore 2 (NPCE) labelled one, which indicates that the fragment size decreases faster for the domain I indicating that digestion starts from the domain I. We have also performed reverse-phase high-performance liquid chromatography (RP-HPLC) to cross-check the findings, supporting our results.

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

Several techniques enable the measurement of digested fragments after proteolysis like mass spectrometry, HPLC, etc. The uniqueness of the proposed approach is its ability to tell directionality using a minimum amount of protein. FCS is sensitive and can be successfully used for picomolar to nanomolar sample concentrations. This is a new application of the single-molecule research that can be used to understand proteins’ digestion profile.

“[…] I see you are now able to think of novel ideas and design the experiments; your training has been completed”

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

There is more than one Eureka moment associated with this specific work. This was the last project I worked on during my PhD. When I conceived the idea and shared with my boss, he said, “I see you are now able to think of novel ideas and design the experiments; your training has been completed”. That was some words of motivation before I could start the next venture. When I started working, I was unsure if I would see any difference in the digestion profile monitored by two different fluorophores labelled at two other locations. If there was no difference, there was no specific conclusion, and it wouldn’t have been as fascinating as the current finding. So when I observed a difference in the digestion profile, I was pretty excited, which was definitely the second eureka moment. The third eureka moment would be when the reverse phase HPLC supported the single-molecule measurements. That was the moment when we found proof that whatever we see in single-molecule measurement is valid.

What do you hope to do next?

Regarding the piece of work, I talked about, I am not following up on that anymore. We had several ideas of trying the technique on different proteins and used different enzymes and different environments. Also, to use drugs to see if they affect the digestion profile etc. But unfortunately, I had to leave to join a postdoc and couldn’t continue with it. If someday I get a chance, I would like to work on the extended ideas. 

Where do you seek scientific inspiration?

The best (or the worst) thing being a researcher is that I don’t know what waits ahead, and from my early childhood, I have felt tremendous attraction towards what is unknown. It feels exciting to delve into the strange world, which is why I keep loving science. Besides, I am blessed to have supervisors (PhD and postdoc) who motivate and have always believed in my ability and dedication. The excitement of discovering something new is often inexplicable. All these factors keep me going, and I wish to be roaming around the field of science forever.

How do you intend to help Indian science improve?

Honestly, “improving Indian science” is a huge goal, and I don’t think I have reached the stage to think about it. Right now, I am doing postdoctoral research and wish to set up my lab in India someday. Provided I can do that; my next goal would be to design experiments with minimum budget and resources possible given the poor condition of the academic budget in India. The next target would be to equip the lab with efficient and budget-friendly instruments. Once all these are in place, one can use single-molecule experiments for several biological and chemical applications using a minimum sample amount. This will cut down the cost of buying or producing expensive samples again and again.

Reference

Sengupta B, Das N, Singh V, Thakur AK, Sen P. Fluorescence correlation spectroscopy as a tool to investigate the directionality of proteolysis. Int. J. Biol. Macromol. 2020; 164: 2524-2534.

Email: bhaswati1487@gmail.com

Job opening in Biocon Biologics

BIOCON Biologics is Hiring.

Department: Biosimilars – MSAT (Downstream)

Experience: 7-12 years

Qualification: MSc/MTech/PhD

Responsibilities:

  1. To manage MSAT Lab functions on routine basis for Downstream related activities.
  2. To ensure product life cycle management.
  3. To handle continuous verification of process, deviation and out of specifications.
  4. To evaluate the alternate vendors for critical raw materials and consumables as required.
  5. Contribute at team discussions of CMC and other decision-making forums for development, clinical and commercial manufacturing.
  6. Review Technology transfer of manufacturing process to CMOs (outside parent site).
  7. Scientific knowledge on microbial downstream processes.

Interested candidates with relevant experience may share profile at sushmitha.101@biocon.com.

Job opening in Kemwell Biopharma

Kemwell Biopharma is hiring experienced professional for Bangalore location.

Position: Executive, Quality control (QC)

Skills required: Experience in in-vitro Bioassays and potency determining ELISA

Experience: 1-3 years

Requirement: Immediate

If you are interested, please share your updated resume to debatree.chakrabarty@kemwellpharma.com

Job openings in CuraTeQ Biologics

Six job openings in CuraTeQ Biologics – Hyderabad.

Skill required: Cell culture, purification and analytical sciences functions.

Qualification: PhD in relevant study area, pursued post-doctoral research for a period of at least 2 years and/or 2-3 years of post- PhD industry work. Overseas PhDs from top 30 Universities (QS Ranking) can apply without post- doctoral experience.

If you are interested, please share your updated resume to HR Nobby Paul (nobby.paul@curateqbio.com).