Research Highlights

Targeting intracellular Salmonella infection using lipid coated nanoparticles

Salmonella is the causative organism of gastroenteritis, typhoid and paratyphoid diseases and is responsible for 2 million fatalities throughout the world. Salmonella has developed ingenious methods to survive even in detrimental intracellular environment of macrophages. BSSE student Rajeev Mudakavi et al. have developed lipid coated mesoporous silica nanoparticle (L-MSN) which are able to target intracellular Salmonella and eliminate them from their host cell by delivering antibiotic directly into its intracellular niche. They also observed that a lower dose of antibiotic encapsulated into L-MSN was sufficient to clear the Salmonella infection from mice. More information of this can be obtained from the following publication.

Publication: R. Mudakavi, A. M. Raichur, and D. Chakravortty , "Lipid coated mesoporous silica nanoparticle as oral delivery system for targeting and treatment of intravacuolar Salmonella infection", RSC Adv., 2014, 4, 61160-61166. DOI: 10.1039/C4RA12973C


Incubator-compatible Perfusion Bioreactor Array

BSSE PhD student Sreenath Balakrishnan and others have designed and built a perfusion bioreactor array that can be used inside a standard CO2 incubator. Each bioreactor has its own custom-made perstaltic pump, whose flow-rate can be adjusted as desired with its integrated electronics. A standard coverslip can be placed inside the biorector with or without scaffold to culture cells. This allows for live imaging and other assays. More information of this can be obtained from the following publication.

Publication: S. Balakrishnan, M. S. Suma, S. R. Raju, S. D. B. Bhargav, S. Arunima, S. Das and G. K. Ananthasuresh, "A Scalable Perfusion Culture System with Miniature Peristaltic Pumps for Live-Cell Imaging Assays with Provision for Microfabricated Scaffolds", BioRes Open Access, 4(1), 343–357. DOI: 10.1089/biores.2015.0024


Pore-forming toxins on artificial membranes

Pore-forming toxins are produced by pathogenic bacteria and punch holes in cell membranes, thereby killing the cell. These toxins are proteins that are unique in their ability to bind to the cell membrane and form pores that are comprised of multiple stands of the same protein. Research has shown which region of the ClyA toxin is important for pore formation, and molecular dynamics simulations have revealed the molecular details that underlie pore formation and membrane interaction.

Publication: M. S. Vaidyanathan, P. Sathyanarayana, P. K. Maiti, S. S. Visweswariah and K. G. Ayappa, "Lysis dynamics and membrane oligomerization pathways for Cytolysin A (ClyA) pore-forming toxin", RSC Adv., 2014, 4, 4930-4942. DOI: 10.1039/C3RA45159C

Synthesis of biodegradable, crosslinked polyesters

This work encompasses the synthesis of an array of biodegradable, crosslinked polyesters with independently tailored degradation, mechanical, release and bioactive properties for biomedical applications. Diacids and polyols endogenous to the body are used as precursors to minimize immune responses to degradation products. The motivation of the present work is to develop a combinatorial strategy to synthesize a library of polymers where synthesis parameters can control various polymer properties independently. Furthermore, it is important to impart bioactivity to the polymers to reduce immunogenicity and susceptibility for microbial infection. Thus, the objective is to chemically incorporate salicylates (salicylic acid and p-aminosalicylic acid) and a benzofuran derivative (usnic acid) by esterification onto the polyester backbone to ensure pharmacological activity. Unlike conventional entrapment of drugs in delivery vehicles, this chemical incorporation allows higher loading, better processability and controlled release.

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Novel biomedical applications for shockwaves


Shockwaves are generated whenever any wave travels faster than the local speed of sound. While shockwaves have been extensively used for various engineering applications, their biological utility remains relatively unexplored. Till date, lithotripsy (using shockwaves to disintegrate kidney stones) is the only application of shockwaves that has been translated to clinics. Research by BSSE student Akshay Datey is aimed at exploring the various effects shockwaves can have on different biological systems. A recent study by Prof. Chakravortty and Prof. Jagadeesh's lab highlighted the potential of shockwaves in disrupting biofilms formed on urinary catheters by three different bacteria, Salmonella, Pseudomonas and Staphylococcus species.  The studies were also extended to a Pseudomonas chronic pneumonia lung infection and Staphylococcus skin suture infection model in mice. Biofilm infections in mice, treated with shock waves became susceptible to antibiotics, unlike untreated biofilms, demonstrating that shock waves, combined with antibiotic treatment can be efficient in treating biofilm infections on medical devices as well as in situ infections. In another study, Datey et al. have used micro-shockwaves along with commercially available desensitizing toothpastes to treat hypersensitivity. This method has shown promising results and can be used in clinics as it strongly blocks dentinal tubules making them resistant to erosion even by acid challenge, unlike conventionally used techniques.

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In vitro model for breast cancer metastasis

Breast cancer is the most common cause of cancer and its metastasis accounts for about 90% of mortality in women. Metastasis is a multi-step process wherein the cancer cells migrate from the primary tumor into another site and establish a tumor. Understanding the molecular mechanisms that orchestrate metastasis provide useful insights into developing therapeutics that prevent or treat metastasis. While conventional two-dimensional (2D) cell culture systems fail to mimic in vivo signaling due to the planar architecture and inappropriate substrate stiffness, animal based xenograft models are expensive and time consuming. To overcome these drawbacks, three dimensional (3D) scaffold based models have been developed which help recapitulate in vivo tissue micro-environment. In this study, for the first time, we have developed a comprehensive Polycaprolactone (PCL) based 3D model to study the events associated with breast cancer metastasis. Using tissue engineering techniques we fabricated 3D scaffolds of polycaprolactone (PCL), with porous geometry and of elastic modulus that was close to that of metastatic breast tumor. Cells cultured in scaffolds grew in multiple layers forming 3D contacts and networks in contrast to cells cultured on 2D dishes, which remained flat with spread morphology. The cells over prolonged culture gave rise to tumoroids. Gene expression and functional analyses showed that the scaffold-cultured cells presented greater EMT, invasive and stemness potential which are critical for manifold events of metastasis like initiation, progression and colonization. Also the scaffold-cultured cells had improved metastatic potential in vivo. Thus these results indicate that culturing breast cancer cells in tissue-like 3D matrices improves their metastatic potential and could hence serve as a better model for metastasis and cancer related inflammation.

Publication:
G.M. Balachander, S.A. Balaji, A. Rangarajan, K. Chatterjee “Enhanced metastatic potential in a 3D tissue scaffold toward a comprehensive in vitro model for breast cancer metastasis” , ACS Applied Materials and Interfaces, 2015 DOI: 10.1021/acsami.5b09064


The role of variability in motor learning

BSSE student Puneet Singh and team explore whether motor variability, often unwanted characteristic of motor performance has any significance in motor learning. They propose that motor variability has two components one caused by redundancy and other is random noise. In this work, Singh et al. quantify redundancy space and investigate its significance and effect on motor learning. They propose that a larger redundancy space leads to faster learning across subjects and the redundant component of motor variability is not noise. They also tested this hypothesis in neurologically diseased conditions to get a mechanistic understanding of how reward-based learning and error-based learning interact and how such learning is affected by redundancy space.

Publication:
P. Singh, S. Jana, A. Ghosal, A. Murthy "Exploration of joint redundancy but not task space variability facilitates supervised motor learning", PNAS, 2016 DOI: 10.1073/pnas.1613383113


The Curious Case of Dynein: Regulation by Myosin I

Molecular motors are tiny machines that convert the energy from the breakdown of ATP to mechanical work and hence bring about changes in the organisation of cellular components, including cargo, organelles and nuclear material. There are three major classes of motor proteins that have been identified: (i) myosins, which are associated with cellular polymers called filamentous actin, and (ii) kinesins and (iii) cytoplasmic dynein, which are associated with pipe-like structures called microtubules. Cell division requires the precise orchestration of a number of proteins in the cell, primarily the microtubules and molecular motors. Cytoplasmic dynein is especially important during cell division for ensuring that the nuclear material is organised properly. In this study, we have uncovered that the localisation and activity of dynein during meiotic cell division in fission yeast is dependent on myosin I. This discovery highlights one of the few examples of interplay between actin-based and microtubule-based motors inside the cell. Studies such as these are important for understanding division of not just healthy cells, but also in cases when the cell division machinery goes rogue, such as during cancer.

Publication:
J. M. Thankachan, S. S. Nuthalapati, N. A. Tirumala, V. Ananthanarayanan "Fission yeast myosin I facilitates PI(4,5)P2–mediated anchoring of cytoplasmic dynein to the cortex", PNAS, 2017 DOI: 10.1073/pnas.pnas.1615883114


Culturing murine neonatal cardiomyocytes

In the current study, our aim was to develop a facile, efficient and cost-effective protocol for culturing the neonatal cardiomyocytes using keratin derived from human hair, which in turn could be used for investigating cardiac hypertrophy in vitro. The nanoscale coating with keratin was characterized using SEM and AFM. Our optimized protocol for culturing of cardiomy- ocytes yielded atleast ~106 cells per heart. The characterization of cardiomyocytes with specific markers revealed that they can attach, grow and show spontaneous contraction on keratin-coated substrates similar to fibronectin-coated surfaces. Cardiomyocyte differentiation was assessed by immunofluorescence. Cardiac hypertrophy was induced using Phenylephrine (PE). Effect of PE-induced hypertrophy on signalling pathways was analysed using western blotting. Signaling proteins such as p-Akt, p-ERK, p-mTOR were up-regulated along with marked increase in protein synthesis on development of hypertrophy. Up-regulation of transcript levels of genes associated with hypertrophy was observed in qRT-PCR. The transient intracellular calcium spikes in beating cardiomyocytes were clearly detected using the fluorescent dye, Fluo-4. We conclude that cardiomyocytes grown on such keratin-coated surfaces are capable of developing the characteristic features of hypertrophy and hence serve as an inexpensive alternative and candidate model for understanding heart failure. Our culture protocol can be used interchangeably for both neonatal rats and mice to study cardiac hypertrophy in vitro. .

Publication:
A. Jain, V. Ravi, J. Muhamed, K. Chatterjee, N. R. Sundaresan, "A simplified protocol for culture of murine neonatal cardiomyocytes on nanoscale keratin coated surfaces", International Journal of Cardiology, 2017 DOI: 10.1016/j.ijcard.2017.01.036