Me working as Associate Professor of Pharmacology at Sri Ramachandra Medical College & Research Institute, Sri Ramachandra Institute of Higher Education & Research, Chennai. I graduated from Mahatma Gandhi Medical College and Research Institute, Pondicherry. Subsequently I pursued my post graduate studies in Pharmacology from SRMC & RI, Chennai. I have secured Diploma in Clinical research from IBI Biosolutions Pvt. Ltd, PG Diploma course in Bioethics from WMA, International Certificate course on the Principles in Bioethics and Human Rights Course & Advance Course in Medical Education by NMC. Currently pursuing MSc in Bioethics under Department of Education, the International Chair in Bioethics, Haifa. At present serving as Member of Health Professional Education, Faculty Development Program, Antiragging, Institutional Program Implementation Unit & Bioethics, AETCOM Committee at SRIHER. Have served as Clinical Pharmacologist & Member of Pharmacy & Therapeutic Committee, SRH for a tenure of 5 Years. Served as Member of Medical Education Unit for 5 years. Currently Serving as CPCSEA Nominee of Govt of India. Serving as Member of Institutional Human Ethics Committee at National Institute of Epidemiology (ICMR) & Meenakshi Academy of Higher Education & Research. Serving as Animal Ethics Committee member for various institutions in Chennai. Served as resource person in various CME’s and Workshops across India. Served as Organizing secretary & has been a part of Organizing committee for several Conferences, Workshops & CMEs at Sri Ramachandra Institute of Higher Education & Research. Been as Judge for various competitions conducted at Sri Ramachandra Institute of Higher Education & Research & various medical colleges in Chennai. Have been a part of list of examiners in several universities across India. Have 27 research paper publications in reputed International and National Journals. Serving as Guide, Co-guide & RAC member for PhD, ISMR-STS studentship & Chancellor summer fellowship programs.
The project aims to assess the early and intermediate effect of PPIs on serum magnesium level in twelve weeks of treatment.
To assess the occurrence of clinical and subclinical hypomagnesemia with PPIs therapy
To assess the associated risk factors of PPIs induced hypomagnesaemia and its significance (Relative Risk -Odds ratio)
Background:
Metformin, a widely used biguanide derivative, operates via various signaling pathways, offering multiple therapeutic advantages. It is the most frequently prescribed medication for Type II Diabetes Mellitus worldwide. By inhibiting gluconeogenic genes through either AMPK-dependent or independent pathways, this pharmaceutical reduces hepatic glucose production [7], yielding an antidiabetic effect. Metformin also suppresses mTOR signaling, stimulates p53, autophagy, and apoptosis [8], and diminishes ROS, DNA damage [2], and inflammation, which contributes to the inhibition of cancer growth [10]. Furthermore, by activating the AMPK pathway or inhibiting mitochondrial complex I, it can modify cellular metabolism [16], thereby alleviating the impacts of conditions such as obesity [6], cardiovascular disease [9], liver disease [3], renal diseases [12], and more.
Review of Scientific Literature
Prior in vivo research has demonstrated that metformin inhibits mTOR activity through an AMPK-dependent mechanism [8], which leads to increased elimination and a reduction in the accumulation of extracellular Aβ [5]. Accordingly, this process enhances the efficacy of autophagy and enhances the engulfment of Aβ clusters by the lysosomal system of microglial cells situated at the blood-brain barrier. Specifically, within this system, MAP1LC3B-II, a microtubule-associated protein that is essential for autophagy substrate protein, interacts with OPTN/optineurin, an autophagy receptor [4]. This interaction recruits Aβ into the autophagic vacuoles. Autophagy is initiated by the STK11/LKB1-PRKAA1/AMPKα pathway following the formation of autophagosomes [11], resulting in the degradation and removal of Aβ plaques from the extracellular environment.
Justification
In Alzheimer’s disease patients, the pathophysiological findings are the result of the widespread loss of neurons and synapses in critical memory storage areas, which is caused by the
accumulation of Aβ plaques. The pathological accumulation of Aβ can be reduced by metformin by increasing the adaptor protein interaction between MAP1LC3B through LC3-interacting motifs and Optineurin, thereby preserving the viability of neuronal cells [5]. Synaptic activity and homeostasis are efficiently regulated throughout the brain [13], including the hippocampus and entorhinal cortex, as a consequence of the increased neuronal cell viability. Consequently, the potential mitigation of AD-related symptoms is presented.
Significance
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder, accounting for approximately 60-70% of all dementia cases. Currently, Alzheimer’s disease (AD) and other dementias associated with aging impact more than 55 million people globally [16] and is projected to affect 152 million by the year 2050 [14]. More than 60% of these individuals are situated in low- and middle-income countries[16], like India, where there were approximately 3.69 million active cases reported, with a prevalence rate of 4.3% [14].
Scientific Lacunae:
Currently, there is a lack of research on microglial autophagy and its role in the extracellular degradation and clearance of Aβ in AD. As a result, this has theoretical implications for understanding the role of microglial cells in maintaining synaptic equilibrium, which is frequently disrupted in Alzheimer’s disease.
Aim:
The aim of this in vitro study is to investigate the impact of metformin on the induction of auto phagocytic processes in the SH-SY5Y cell line, which serves as a model for neurodegenerative disorders like Alzheimer’s Disease [1], that are essential for the extracellular clearance of Aβ plaques.
OBJECTIVES:
• Primary Objective:
o To evaluate the level of STK11/LKB1-PRKAA1/AMPKα pathway activity
• Secondary Objectives:
o To assess the interaction between MAP1LC3B and Optineurin in microglia
o To measure the degree of autophagy in the microglia of Aβ plaques
Proposed Hypotheses:
(1) The STK11/LKB1-PRKAA1/AMPKα pathway activity will be enhanced by the administration of metformin therapy.
(2) The implementation of metformin therapy will result in an improved interaction between Optineurin and MAP1LC3B in microglia, which will result in an increased sequestration of Aβ plaques by autophagosomes.
METHODOLOGY:
This study will be conducted in the clinical laboratory of Department of Pharmacology in
SRIHER.
TYPE OF STUDY: In Vitro Study
PLACE OF STUDY: Sri Ramachandra Medical College and Research Institute
CELL LINE: SH-SY5Y cell line
PROCEDURE:
Cell culture
The SH-SY5Y cell line will be cultivated in 100-mm dishes using Dulbecco’s modified Eagle’s medium.
Western blot analysis
Western blots will be carried out under appropriate denaturing and reducing conditions. Blots will be incubated for 16 hours at 4°C with antibodies against Aβ, MAP1LC3B, Ser428-phosphorylated STK11, Thr172-phosphorylated PRKAA1, and anti-PRKAA1. The bands will be densitometrically quantified using Image J.
Protein–protein interaction assay
The Proximity Ligation Assay detection reagents will be used to evaluate the interactions between Aβ peptides and autophagosomes. SH-SY5Y cells will be cultivated on glass coverslips and then treated with a solution of 4% paraformaldehyde and 0.1 M phosphate buffer at room temperature for 30 minutes. The cell membranes will be made permeable by incubating them in a solution of 0.05 M Tris buffer, pH 7.4, containing 0.1% Triton X-100, 2% bovine serum
albumin, and 2% normal horse serum for 30 minutes. The cultures will be maintained at a temperature of 4 °C overnight in a solution containing anti-MAP1LC3B antibodies. Following, they will be appropriately washed and incubated in a solution containing PLA probes for 1 hour at a temperature of 37 °C. The cultures will then be rinsed and then exposed to the Ligation-Ligase solution for a duration of 30 minutes at a temperature of 37 °C. Subsequently, they will be incubated with the amplification-polymerase solution for a period of 100 minutes at the same temperature. Afterwards, the culture will be prepared with DAPI and examined using an electron microscope.
Immunohistochemistry
The SH-SY5Y cells will be made permeable by exposing them to a solution consisting of 0.05 M Tris buffer, pH 7.4, with 0.1% Triton X-100, 2% bovine serum albumin, and 2% normal horse serum for a duration of 30 minutes. The sections will be placed in a solution containing anti-Aβ antibodies and kept at a temperature of 4 °C overnight. Following, they will be washed and incubated with secondary antiserum, which consists of fluorescein isothiocyanate-conjugated antibodies and cyanine 3 fluorescent-conjugated antibodies, at a dilution of 1:500. Cells were stained with DAPI and mounted. Afterwards these cells will be captured using a confocal microscope and subsequently analyzed.
CONFIDENTIALITY AND ETHICAL CLEARANCE
The Institutional Ethics Committee will be notified to obtain approval for conducting this in vitro study. An ethical clearance waiver will be applied and procured for this in vitro study. Procedures will only then be carried out after obtaining consent and ensured that they are performed in a sterile and appropriate manner.
IMPLICATIONS
The purpose of this study is to see if there is a significant reduction in the accumulation of Aβ given an increased extracellular clearance of these Aβ clusters after the administration of metformin.
• Implementation in individuals who are at a high risk of developing Alzheimer’s may pose the beneficial effect of significant delay in disease pathology
• Individuals currently experiencing the effects of cognitive deterioration due to Alzheimer’s disease may potentially experience a substantial reduction in disease symptoms through administration.
By incorporating this commonly used biguanide derivative, significant positive outcomes can be achieved in the management of Alzheimer’s disease by targeting the underlying pathophysiological processes. This, in turn, can help maintain the strength and viability of neuronal synapses in various regions of the brain thus lessening cognitive deterioration.
Significance:
Parkinson’s disease (PD) ranks as the second most prevalent neurodegenerative condition globally, impacting approximately 10 million individuals, with around 1% of those aged over 60 being affected. Projections suggest that the prevalence of Parkinson’s could rise to affect 17 million individuals by 2040, constituting roughly 22% of the worldwide population aged 60 and above by 2050 [3]
Background:
Due to its anti-inflammatory properties, bee venom is an effective treatment for a wide range of conditions. These properties have made it a useful tool in alleviation of chronic inflammatory ailments like rheumatoid arthritis, cancer, and chronic pain in back pain [5]. The contributing factors to these actions are bioactive peptides, including mast cell degranulation peptides, melittin, apamin, and adolapin [4].
Review of Scientific Literature:
Previous scientific studies have indicated that the bee venom peptide apamin functions as an allosteric inhibitor of Ca2+ activated K+ (SK) channels, which has been observed to regulate dopamine neuron activity [6]. The SK voltage independent channels are comprised of three distinct types: SK1, SK2, and SK3, which are distributed throughout the body, including the brain, heart, and skeletal muscle, among others. Situated in dopaminergic neurons within the brainstem, neocortex, cerebellum, and hypothalamus, these SK are localized in significant quantities [1]. Blocking these SK channels causes a transient change in membrane potential due to K+ efflux, which works in tandem with increasing intracellular calcium concentration. Specifically, apamin has been shown to target SK2 and SK3 giving rise to structural excitatory changes of dopamine neurons indicative of its neuroprotective potential in neurodegenerative diseases [6].
JUSTIFICATION:
Due to a significant reduction in the stimulating electrical activity of dopaminergic neurons, which is a characteristic of Parkinson’s disease, the mesencephalic dopaminergic neurons undergo a gradual process of cell death. Apamin administration can potentially rescue mesencephalic neurons in the substantia nigra with a low pacemaker frequency in Parkinson’s disease by inducing excitatory structural changes such as higher extracellular [K+]o and higher intracellular [Ca2+]o thus causing low level [Na+]o conductance [2].
SCIENTFIC LACUNAE:
At present, there is insufficient research to ascertain the extent of the neuroprotective impact on the nigrostriatal dopaminergic neurons situated in the mesencephalon following the
administration of apamin, as well as whether these effects can be enhanced by existing drugs for Parkinson’s disease.
AIM:
This comparative in vitro study aims to investigate the disparity in excitatory electrical alterations in the SH-SY5Y cell line [7], which serves as a model for neurodegenerative disorders like Parkinson’s disease.
OBJECTIVES:
•
To determine the level of increase extracellular [K+]o and higher intracellular [Ca2+]o in SH-SY5Y cells after administration of the isolated Bee Venom peptide apamin
•
To determine the measurement of Dopamine uptake in SH-SY5Y after administration of the isolated Bee Venom peptide apamin
METHODOLOGY
This study will be conducted in the clinical laboratory of Department of Pharmacology in SRIHER.
TYPE OF STUDY: In Vitro Study
PLACE OF STUDY: Sri Ramachandra Medical College and Research Institute
STUDY CELL: SH-SY5Y cell line
PROCEDURE:
Measurement of Neurotransmitter uptake:
The SH-SY5Y cell will undergo preincubation with 50 nm [3H]-DA for 10 minutes in 500 μl of PBS solution supplemented with 5 mm glucose and 100 μm ascorbic acid. Following a 15-minute incubation period, the process will be halted by quickly washing the cells twice with cold PBS. Subsequently, the level of [3H] Dopamine will be assessed using micro autoradiography techniques [6].
Measurement of Intracellular Calcium
Cells will be incubated for a duration of 7 days with 10 μm Calcium Green-1-AM for a period of 30 minutes at 37°C. Consequently, these cells will be followed by two washes with serum-free glucose-supplemented N5 medium to eliminate any excess indicator. After a recovery period of 30 minutes these cells will be visualized through immunofluorescent assay techniques [2].
Measurement of Extracellular Potassium
Measurement of potassium will be conducted in similar fashion to calcium however cells will be incubated with GEPII [2].
Statistical Analysis
The quantification of cells will be performed using confocal images obtained through the micro autoradiography technique to measure dopamine levels, as well as immunofluorescent techniques to measure intracellular calcium and extracellular potassium levels. The data will be
imported into the SPSS STATA analytical software for the purpose of conducting a series of T-Tests. The tests will compare cells that have not been treated with apamin to cells that have been treated with apamin. The comparison will be based on [3H] Dopamine uptake, intracellular calcium levels, and extracellular potassium levels.
Awarded most active member UNESCO chair in Bioethics, SRIHER on 12th October 2018
Awarded GATE Research Starter Grants to Young Faculty for “Assessing Serum Magnesium Concentration Among Patients Receiving Proton Pump Inhibitors (PPI’s)”
Won the Employee of the year 2017 for Pharmacology in Employee Appreciation week-2017, SRMC.
Won the Best Poster Award for paper titled In Vitro Free Radical Scavenging activity & Alpha amylase inhibitory activity of Pterocarpus marsupium extract at ISLETCON-2015 held on 27.07.2015 to 29.07.2015 in Sree Balaji Medical College & Hospital, Chennai
Accredited National Training Faculty for 3T-IBHSc Course, UNESCO Chair in Bioethics Haifa
Nominee of CPCSEA, Government of India
As been recognized as Best Teacher in Pharmacology, SRIHER and was asked to provide valuable inputs towards improvement in medical education in the meeting held on 7th Feb 2014.
Chaired a session on “Research Protocol Writing” at Saveetha Medcial College, Thandalam, Chennai
Served as Panelist in Panel discussion for World Bioethics Day Celebration on 26th October.
Awarded Teaching Excellence Award-2020 by Department of Health Professional Education, SRIHER
Member of Faculty Development Program at SRMC
Member of International Network of the UNESCO chair in Bioethics at SRIHER
Member of Department of Health Profession Education at SRIHER
Member of Online Education Committee at SRIHER
Member of Blended learning Committee at SRIHER
Member of Antiragging squad at SRIHER
Member of Mentoring Team for MBBS students
Member of Institutional Program Implementation Unit (IPIU)