Subhathirai Subramaniyan Parimalam

Bio: Subhathirai Subramaniyan Parimalam is an academic researcher from Concordia University. The author has contributed to research in topics: Tau protein & Shear stress. The author has an hindex of 3, co-authored 8 publications receiving 23 citations. Previous affiliations of Subhathirai Subramaniyan Parimalam include Kyoto University & Concordia University Wisconsin.

On-chip microtubule gliding assay for parallel measurement of tau protein species.

Abstract: Tau protein is a well-established biomarker for a group of neurodegenerative diseases collectively called tauopathies. So far, clinically relevant detection of tau species in cerebrospinal fluid (CSF) cannot be achieved without immunological methods. Recently, it was shown that different tau isoforms including the ones carrying various types of mutations affect microtubule (MT)–kinesin binding and velocity in an isoform specific manner. Here, based on these observations, we developed a microfluidic device to analyze tau mutations, isoforms and their ratios. The assay device consists of three regions: a MT reservoir which captures MTs from a solution to a kinesin-coated surface, a microchannel which guides gliding MTs, and an arrowhead-shaped collector which concentrates MTs. Tau-bound fluorescently labeled MTs (tau-MTs) were assayed, and the increase in fluorescence intensity (FI) corresponding to the total number of MTs accumulated was measured at the collector. We show that our device is capable of differentiating 3R and 4R tau isoform ratios and effects of point mutations within 5 minutes. Furthermore, radially oriented collector regions enable simultaneous FI measurements for six independent assays. Performing parallel assays in the proposed device with minimal image processing provides a cost-efficient, easy-to-use and fast tau detection platform.

Lab-On-A-Chip for the Development of Pro-/Anti-Angiogenic Nanomedicines to Treat Brain Diseases

Abstract: There is a huge demand for pro-/anti-angiogenic nanomedicines to treat conditions such as ischemic strokes, brain tumors, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Nanomedicines are therapeutic particles in the size range of 10–1000 nm, where the drug is encapsulated into nano-capsules or adsorbed onto nano-scaffolds. They have good blood–brain barrier permeability, stability and shelf life, and able to rapidly target different sites in the brain. However, the relationship between the nanomedicines’ physical and chemical properties and its ability to travel across the brain remains incompletely understood. The main challenge is the lack of a reliable drug testing model for brain angiogenesis. Recently, microfluidic platforms (known as “lab-on-a-chip” or LOCs) have been developed to mimic the brain micro-vasculature related events, such as vasculogenesis, angiogenesis, inflammation, etc. The LOCs are able to closely replicate the dynamic conditions of the human brain and could be reliable platforms for drug screening applications. There are still many technical difficulties in establishing uniform and reproducible conditions, mainly due to the extreme complexity of the human brain. In this paper, we review the prospective of LOCs in the development of nanomedicines for brain angiogenesis–related conditions.

A Narrative review of scientific validation of gold- and silver-based Indian medicines and their future scope

Abstract: Metals are incinerated along with plant extracts and used as oral drugs in the Indian traditional medicines, such as Ayurveda and Siddha. Gold and silver ashes are predominantly used in cancer therapy and for treating neuronal disorders. Since the beginning of the nano-era, these ashes were investigated for their characteristics, especially the size of the particles. Numerous hypotheses were advanced to repurpose them as nanomedicines. During the last two decades, several studies were conducted studying the correlation of the particle size and the therapeutic effects. Here, we discuss the processes used to prepare gold and silver ash that result in the formation of nanoand micro-scale particles. Further, we review recent works on the ashes using modern tools and their scope, in nanomedicine. We emphasize the need to generate experimental data of high-quality to reinforce the scientific validation of these traditional medicines.

Microtubule density and landing rate as parameters to analyze tau protein in the MT-kinesin "gliding" assay

Abstract: Microtubule-associated protein (MAP) tau is a well-established hallmark of a large group of age related neurodegenerative diseases collectively called tauopathies. Under pathological conditions the equilibrium of tau binding to the MTs is perturbed, either by misregulation in the expression levels of specific tau isoforms or by MAPT gene mutations. Preclinical detection of such misregulated tau proteins in cerebrospinal fluid (CSF) is desirable for differential diagnosis and effective prognosis of neurodegeneration. Conventional tau protein detection methods utilize tau isoform-specific antibodies. Such immuno-based protocols, including enzyme-linked immunosorbent assay (ELISA) and Western blots have appropriate sensitivity and specificity, but often show high variability and are time consuming. Here, we established a non-immuno tau protein detection method utilizing microtubule (MT)-kinesin “gliding”assay. All the six tau isoforms expressed in the human brain (0N3R, 1N3R, 2N3R, 0N4R, 1N4R and 2N4R) and five MAPT gene mutants (V248L, G272V, P301L, V337M and R406W) were studied. The landing rate, binding density and gliding velocity of MTs with respect to each tau type were determined and are proposed as tau detection parameters. The detection parameters depicted the type of tau bound to the MTs. Furthermore, MT landing rate and density were found to be superior to gliding velocity in differentiating tau isoforms and mutants. The 3R vs. 4R isoforms, their admixtures, wild vs. mutant 2N4R and specific mutants were differentiated. Our data show that MT-kinesin gliding assay provides a convenient, lab-on-a-chip (LOC) compatible and antibody-free protocol for tau protein analysis.

Study of Incinerated Silver Used in Indian Traditional Medicine Systems

Abstract: Incinerated metal particles such as gold, silver, copper and iron are administered as internal medicines in the Indian systems of medicine, Siddha and Ayurveda, respectively. We studied the structure, composition and properties of the Incinerated Silver (IAg) particles (Veli parpam/Rajata bhasma) as well as their spectra and localisation in cancer cells (HeLa, A549 and HCT 116). The average crystallite size, particle size, and hydrodynamic diameter were found to be 54.6 nm, 300 nm to 1.7 m, and 530 nm-1.1 m, respectively. The particles have a negative surface charge with a zeta potential of -22.87±1.34 mV. The IAg particles are made up of Ag, Ca, Fe, Hg and other trace elements. Further, their subcellular properties and locations were analysed in cancer cell lines. The preliminary results obtained from the SEM and hyperspectral imaging show the uptake of the IAg particles and aggregates.

Perfused 3D angiogenic sprouting in a high-throughput in vitro platform

Abstract: Angiogenic sprouting, the growth of new blood vessels from pre-existing vessels, is orchestrated by cues from within the cellular microenvironment, such as biochemical gradients and perfusion However, many of these cues are missing in current in vitro models of angiogenic sprouting We here describe an in vitro platform that integrates both perfusion and the generation of stable biomolecular gradients and demonstrate its potential to study more physiologically relevant angiogenic sprouting and microvascular stabilization The platform consists of an array of 40 individually addressable microfluidic units that enable the culture of perfused microvessels against a three-dimensional collagen-1 matrix Upon the introduction of a gradient of pro-angiogenic factors, the endothelial cells differentiated into tip cells that invaded the matrix Continuous exposure resulted in continuous migration and the formation of lumen by stalk cells A combination of vascular endothelial growth factor-165 (VEGF-165), phorbol 12-myristate 13-acetate (PMA), and sphingosine-1-phosphate (S1P) was the most optimal cocktail to trigger robust, directional angiogenesis with S1P being crucial for guidance and repetitive sprout formation Prolonged exposure forces the angiogenic sprouts to anastomose through the collagen to the other channel This resulted in remodeling of the angiogenic sprouts within the collagen: angiogenic sprouts that anastomosed with the other perfusion channel remained stable, while those who did not retracted and degraded Furthermore, perfusion with 150 kDa FITC-Dextran revealed that while the angiogenic sprouts were initially leaky, once they fully crossed the collagen lane they became leak tight This demonstrates that once anastomosis occurred, the sprouts matured and suggests that perfusion can act as an important survival and stabilization factor for the angiogenic microvessels The robustness of this platform in combination with the possibility to include a more physiological relevant three-dimensional microenvironment makes our platform uniquely suited to study angiogenesis in vitro

The model of local axon homeostasis - explaining the role and regulation of microtubule bundles in axon maintenance and pathology

Abstract: Axons are the slender, cable-like, up to meter-long projections of neurons that electrically wire our brains and bodies. In spite of their challenging morphology, they usually need to be maintained for an organism's lifetime. This makes them key lesion sites in pathological processes of ageing, injury and neurodegeneration. The morphology and physiology of axons crucially depends on the parallel bundles of microtubules (MTs), running all along to serve as their structural backbones and highways for life-sustaining cargo transport and organelle dynamics. Understanding how these bundles are formed and then maintained will provide important explanations for axon biology and pathology. Currently, much is known about MTs and the proteins that bind and regulate them, but very little about how these factors functionally integrate to regulate axon biology. As an attempt to bridge between molecular mechanisms and their cellular relevance, we explain here the model of local axon homeostasis, based on our own experiments in Drosophila and published data primarily from vertebrates/mammals as well as C. elegans. The model proposes that (1) the physical forces imposed by motor protein-driven transport and dynamics in the confined axonal space, are a life-sustaining necessity, but pose a strong bias for MT bundles to become disorganised. (2) To counterbalance this risk, MT-binding and -regulating proteins of different classes work together to maintain and protect MT bundles as necessary transport highways. Loss of balance between these two fundamental processes can explain the development of axonopathies, in particular those linking to MT-regulating proteins, motors and transport defects. With this perspective in mind, we hope that more researchers incorporate MTs into their work, thus enhancing our chances of deciphering the complex regulatory networks that underpin axon biology and pathology.

Recent developments in microfluidic technologies for central nervous system targeted studies

Abstract: Neurodegenerative diseases (NDs) bear a lot of weight in public health. By studying the properties of the blood-brain barrier (BBB) and its fundamental interactions with the central nervous system (CNS), it is possible to improve the understanding of the pathological mechanisms behind these disorders and create new and better strategies to improve bioavailability and therapeutic efficiency, such as nanocarriers. Microfluidics is an intersectional field with many applications. Microfluidic systems can be an invaluable tool to accurately simulate the BBB microenvironment, as well as develop, in a reproducible manner, drug delivery systems with well-defined physicochemical characteristics. This review provides an overview of the most recent advances on microfluidic devices for CNS-targeted studies. Firstly, the importance of the BBB will be addressed, and different experimental BBB models will be briefly discussed. Subsequently, microfluidic-integrated BBB models (BBB/brain-on-a-chip) are introduced and the state of the art reviewed, with special emphasis on their use to study NDs. Additionally, the microfluidic preparation of nanocarriers and other compounds for CNS delivery has been covered. The last section focuses on current challenges and future perspectives of microfluidic experimentation.

Ratiometric ATP detection on gliding microtubules based on bioorthogonal fluorescence conjugation

Abstract: An ingenious microtubule functionalization strategy based on the novel bioorthogonal conjugation is developed to construct fluorescent ratiometric probes for ATP sensing. By single excitation, newly synthesized probe RT-1 exhibited green emission (485 nm) from the bioorthogonal fluorescence conjugation part and orange emission (584 nm) from the ATP sensing part. The fluorescence intensity ratio (I584/I485) displayed good linear response in ATP ranges of 0–2.5 mM (y = 0.9282x + 0.2398, R2 = 0.9705, LOD = 0.0354 mM) and 2.0–10.0 mM (y = 2.7153x-3.6234, R2 = 0.9911, LOD = 0.0121 mM). Moreover, a paclitaxel derivate probe RT-2 was developed to functionalize in vitro polymerized microtubule for in situ ATP detection. This simple and convenient strategy is anticipated to stimulate more microtubule related applications in both in vitro and intracellular studies.

The model of local axon homeostasis - explaining the role and regulation of microtubule bundles in axon maintenance and pathology

Abstract: Axons are the slender, cable-like, up to meter-long projections of neurons that electrically wire our brain and body. In spite of their challenging morphology, they usually need to be maintained for an organism’s lifetime. This makes them key lesion sites in pathological processes of ageing, injury and neurodegeneration. To better understand how axons are formed and maintained long-term, we focus here on the parallel bundles of microtubules (MTs) which form their indispensable structural backbones and highways for life-sustaining cargo transport and organelle dynamics. Many MT-binding and -regulating proteins in axons have prominent hereditary links to axon degeneration, but knowing their molecular roles is usually insufficient to explain their roles during axon morphogenesis, maintenance or pathology. Such understanding requires deciphering how these proteins interact in regulatory networks to implement observed cellular phenomena. Here we propose the model of local axon homeostasis as a conceptual framework that attempts to combine current knowledge into one coherent interactome. According to this model, each area of an axon is self-sustaining through local auto-regulatory networks; these networks maintain a balance between (1) the enormous mechanical challenges posed by the life-sustaining intra-axonal motor dynamics and (2) the maintenance activities required to sustain MT bundles as the highways needed for those dynamics. This model offers a new level of explanation, and we hope that it will influence experimental approaches leading to more MT-related experimental data across the spectrum of axonopathies - which are urgently required to decipher the important contributions of MTs to axon biology and pathology.