An Undergraduate Cell Biology Lab: Western Blotting to Detect Proteins from Drosophila Eye

We have developed an undergraduate laboratory to allow detection and localization of proteins in the compound eye of Drosophila melanogaster, a.k.a fruit fly. This lab was a part of the undergraduate curriculum of the cell biology laboratory course aimed to demonstrate the use of Western Blotting technique to study protein localization in the adult eye of Drosophila. Western blotting, a two-day laboratory exercise, can be used to detect the presence of proteins of interests from total protein isolated from a tissue. The first day involves isolation of proteins from the tissue and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel electrophoresis to separate the denatured proteins in accordance to their molecular weight/s. The separated proteins are then transferred to the Nitrocellulose or Polyvinylidene difluoride (PVDF) membrane in an overnight transfer. The second day lab involves detection of proteins (transferred to the membrane) using Ponceau-S stain, followed by immunochemistry to detect the protein of interest along the total protein transferred to the membrane. The presence of our protein of interest is carried out by using a primary antibody against the protein, followed by binding of secondary antibody which is tagged to an enzyme. The protein band can be detected by using the kit, which provides substrate to the enzyme. The protein levels can be quantified, compared, and analyzed by calculating the respective band intensities. Here, we have used fly eyes to detect the difference in level of expression of Tubulin (Tub) and Wingless (Wg) proteins in the adult eye of Drosophila in our class. The idea of this laboratory exercise is to: (a) familiarize students with the underlying principles of protein chemistry and its application to diverse areas of research, (b) to enable students to get a hands-on-experience of this biochemical technique

48784 (GMR17G08)

dpp enhancer - 48784 Id - GMR 17G08 Location 2L:2450278,2451074 Base pairs – 796 b

Exploring the Efficacy of Natural Products in Alleviating Alzheimer’s Disease Using Animal Models

Alzheimer’s disease (hereafter AD) is a progressive neurodegenerative disorder that affects the central nervous system. There are multiple factors that cause AD, viz., accumulation of extracellular Amyloid-beta 42 plaques, intracellular hyper-phosphorylated Tau tangles, generation of reactive oxygen species due to mitochondrial dysfunction and genetic mutations. The plaques and tau tangles trigger aberrant signaling, which eventually cause cell death of the neurons. As a result, there is shrinkage of brain, cognitive defects, behavioral and psychological problems. To date, there is no direct cure for AD. Thus, scientists have been testing various strategies like screening for the small inhibitor molecule library or natural products that may block or prevent onset of AD. Historically, natural products have been used in many cultures for the treatment of various diseases. The research on natural products have gained importance as the active compounds extracted from them have medicinal values with reduced side effects, and they are bioavailable. The natural products may target the proteins or members of signaling pathways that get altered in specific diseases. Many natural products are being tested in various animal model systems for their role as a potential therapeutic target for AD, and to address questions about how these natural products can rescue AD or other neurodegenerative disorders. Some of these products are in clinical trials and results are promising because of their neuroprotective, anti-inflammatory, antioxidant, anti-amyloidogenic, anticholinesterase activities and easy availability. This review summarizes the use of animal model systems to identify natural products, which may serve as potential therapeutic targets for AD

48770 (GMR17E04)

dpp enhancer - 48770 Id - GMR17E04 Location 2L: 2428913..2432834 Base pairs – 3921 b

Unbiased automated quantitation of ROS signals in live retinal neurons of Drosophila using Fiji/ImageJ

Numerous imaging modules are utilized to study changes that occur during cellular processes. Besides qualitative (immunohistochemical) or semiquantitative (Western blot) approaches, direct quantitation method(s) for detecting and analyzing signal intensities for disease(s) biomarkers are lacking. Thus, there is a need to develop method(s) to quantitate specific signals and eliminate noise during live tissue imaging. An increase in reactive oxygen species (ROS) such as superoxide (O2•-) radicals results in oxidative damage of biomolecules, which leads to oxidative stress. This can be detected by dihydroethidium staining in live tissue(s), which does not rely on fixation and helps prevent stress on tissues. However, the signal-to-noise ratio is reduced in live tissue staining. We employ the Drosophila eye model of Alzheimer\u27s disease as a proof of concept to quantitate ROS in live tissue by adapting an unbiased method. The method presented here has a potential application for other live tissue fluorescent images

Osteological morphometric analysis of instrumentation safe zones of C1 and C2 vertebra in North Indian population: a multicentric study

Background: The complex anatomy and critical functional role of the C1 and C2 vertebrae necessitate precise understanding of safe zones for instrumentation to mitigate risks during surgical interventions. This study aimed to conduct a comprehensive morphometric analysis to identify and characterize safe zones for instrumentation within C1 and C2 vertebrae. Though there are multiple radiological based studies, actual osteological measurements are not available for North Indian population. Methods: 200 atlas and axis vertebrae were measured within an accuracy of 0.01 mm to ascertain various dimensions, distances and angles to guide safe exposure and instrumentation. To the best of our knowledge this observational morphometric study is first to provide actual osteological measurements in large number of C1 and C2 vertebrae in North Indian population. Results: The morphometric analysis revealed precise measurements of pedicle dimensions, transverse foramen parameters, and distances from key anatomical landmarks within C1 and C2 vertebrae. Safe zones for instrumentation were identified based on these measurements, considering the optimal implant size and trajectory to minimize the risk of neural or vascular damage. Differences between the morphology of North Indian, South Indian and Turkish C1 and C2 morphology was also identified. Conclusions: This study provides critical insights into the morphometric parameters which can be used to identify safe zones for instrumentation within the C1 and C2 vertebrae. The identified safe zones and associated measurements are essential for optimizing surgical strategies, enhancing instrumentation accuracy, and ultimately improving patient outcomes during craniovertebral surgical procedures. Spine Surgeons can utilize this data to tailor surgical approaches and implant placements, promoting safer and more effective interventions in the challenging anatomical region of the craniovertebral junction

Drosophila Eye Model to Study Neuroprotective Role of CREB Binding Protein (CBP) in Alzheimer’s Disease

Background: The progressive neurodegenerative disorder Alzheimer’s disease (AD) manifests as loss of cognitive functions, and finally leads to death of the affected individual. AD may result from accumulation of amyloid plaques. These amyloid plaques comprising of amyloid-beta 42 (Aβ42) polypeptides results from the improper cleavage of amyloid precursor protein (APP) in the brain. The Aβ42 plaques have been shown to disrupt the normal cellular processes and thereby trigger abnormal signaling which results in the death of neurons. However, the molecular-genetic mechanism(s) responsible for Aβ42 mediated neurodegeneration is yet to be fully understood. Methodology/Principal Findings: We have utilized Gal4/UAS system to develop a transgenic fruit fly model for Aβ42 mediated neurodegeneration. Targeted misexpression of human Aβ42 in the differentiating photoreceptor neurons of the developing eye of transgenic fly triggers neurodegeneration. This progressive neurodegenerative phenotype resembles Alzheimer’s like neuropathology. We identified a histone acetylase, CREB Binding Protein (CBP), as a genetic modifier of Aβ42 mediated neurodegeneration. Targeted misexpression of CBP along with Aβ42 in the differentiating retina can significantly rescue neurodegeneration. We found that gain-of-function of CBP rescues Aβ42 mediated neurodegeneration by blocking cell death. Misexpression of Aβ42 affects the targeting of axons from retina to the brain but misexpression of full length CBP along with Aβ42 can restore this defect. The CBP protein has multiple domains and is known to interact with many different proteins. Our structure function analysis using truncated constructs lacking one or more domains of CBP protein, in transgenic flies revealed that Bromo, HAT and polyglutamine (BHQ) domains together are required for the neuroprotective function of CBP. This BHQ domain of CBP has not been attributed to promote survival in any other neurodegenerative disorders. Conclusions/Significance: We have identified CBP as a genetic modifier of Aβ42 mediated neurodegeneration. Furthermore, we have identified BHQ domain of CBP is responsible for its neuroprotective function. These studies may have significant bearing on our understanding of genetic basis of AD

A Positive Feedback Loop of Hippo- and c-Jun-Amino-Terminal Kinase Signaling Pathways Regulates Amyloid-Beta-Mediated Neurodegeneration

Alzheimer\u27s disease (AD, OMIM: 104300) is an age-related disorder that affects millions of people. One of the underlying causes of AD is generation of hydrophobic amyloid-beta 42 (Aβ42) peptides that accumulate to form amyloid plaques. These plaques induce oxidative stress and aberrant signaling, which result in the death of neurons and other pathologies linked to neurodegeneration. We have developed a Drosophila eye model of AD by targeted misexpression of human Aβ42 in the differentiating retinal neurons, where an accumulation of Aβ42 triggers a characteristic neurodegenerative phenotype. In a forward deficiency screen to look for genetic modifiers, we identified a molecularly defined deficiency, which suppresses Aβ42-mediated neurodegeneration. This deficiency uncovers hippo (hpo) gene, a member of evolutionarily conserved Hippo signaling pathway that regulates growth. Activation of Hippo signaling causes cell death, whereas downregulation of Hippo signaling triggers cell proliferation. We found that Hippo signaling is activated in Aβ42-mediated neurodegeneration. Downregulation of Hippo signaling rescues the Aβ42-mediated neurodegeneration, whereas upregulation of Hippo signaling enhances the Aβ42-mediated neurodegeneration phenotypes. It is known that c-Jun-amino-terminal kinase (JNK) signaling pathway is upregulated in AD. We found that activation of JNK signaling enhances the Aβ42-mediated neurodegeneration, whereas downregulation of JNK signaling rescues the Aβ42-mediated neurodegeneration. We tested the nature of interactions between Hippo signaling and JNK signaling in Aβ42-mediated neurodegeneration using genetic epistasis approach. Our data suggest that Hippo signaling and JNK signaling, two independent signaling pathways, act synergistically upon accumulation of Aβ42 plaques to trigger cell death. Our studies demonstrate a novel role of Hippo signaling pathway in Aβ42-mediated neurodegeneration

Genomic characterization and epidemiology of an emerging SARS-CoV-2 variant in Delhi, India

Delhi, the national capital of India, experienced multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks in 2020 and reached population seropositivity of >50% by 2021. During April 2021, the city became overwhelmed by COVID-19 cases and fatalities, as a new variant, B.1.617.2 (Delta), replaced B.1.1.7 (Alpha). A Bayesian model explains the growth advantage of Delta through a combination of increased transmissibility and reduced sensitivity to immune responses generated against earlier variants (median estimates: 1.5-fold greater transmissibility and 20% reduction in sensitivity). Seropositivity of an employee and family cohort increased from 42% to 87.5% between March and July 2021, with 27% reinfections, as judged by increased antibody concentration after a previous decline. The likely high transmissibility and partial evasion of immunity by the Delta variant contributed to an overwhelming surge in Delhi