Diffuse retro-reflective imaging for improved mosquito tracking around human baited bednets

Robust imaging techniques for tracking insects have been essential tools in numerous laboratory and field studies on pests, beneficial insects and model systems. Recent innovations in optical imaging systems and associated signal processing have enabled detailed characterisation of nocturnal mosquito behaviour around bednets and improvements in bednet design, a global essential for protecting populations against malaria. Nonetheless, there remain challenges around ease of use for large scale in situ recordings and extracting data reliably in the critical areas of the bednet where the optical signal is attenuated. Here we introduce a retro-reflective screen at the back of the measurement volume, which can simultaneously provide diffuse illumination, and remove optical alignment issues whilst requiring only one-sided access to the measurement space. The illumination becomes significantly more uniform, although, noise removal algorithms are needed to reduce the effects of shot noise particularly across low intensity bednet regions. By systematically introducing mosquitoes in front and behind the bednet in lab experiments we are able to demonstrate robust tracking in these challenging areas. Overall, the retro-reflective imaging setup delivers mosquito segmentation rates in excess of 90% compared to less than 70% with back-lit systems

Diffuse retro-reflective imaging for improved video tracking of mosquitoes at human baited bednets

Robust imaging techniques for tracking insects have been essential tools in numerous laboratory and field studies on pests, beneficial insects and model systems. Recent innovations in optical imaging systems and associated signal processing have enabled detailed characterization of nocturnal mosquito behaviour around bednets and improvements in bednet design, a global essential for protecting populations against malaria. Nonetheless, there remain challenges around ease of use for large-scale in situ recordings and extracting data reliably in the critical areas of the bednet where the optical signal is attenuated. Here, we introduce a retro-reflective screen at the back of the measurement volume, which can simultaneously provide diffuse illumination, and remove optical alignment issues while requiring only one-sided access to the measurement space. The illumination becomes significantly more uniform, although noise removal algorithms are needed to reduce the effects of shot noise, particularly across low-intensity bednet regions. By systematically introducing mosquitoes in front of and behind the bednet in laboratory experiments, we are able to demonstrate robust tracking in these challenging areas. Overall, the retro-reflective imaging set-up delivers mosquito segmentation rates in excess of 90% compared to less than 70% with backlit systems

Identification of potential herbal inhibitor of acetylcholinesterase associated alzheimer’s disorders using molecular docking and molecular dynamics simulation

Cholinesterase inhibitors (ChE-Is) are the standard for the therapy of AD associated disorders and are the only class of approved drugs by the Food and Drug Administration (FDA). Additionally, acetylcholinesterase (AChE) is the target for many Alzheimer’s dementia drugs which block the function of AChE but have some side effects. Therefore, in this paper, an attempt was made to elucidate cholinesterase inhibition potential of secondary metabolite from Cannabis plant which has negligible or no side effect. Molecular docking of 500 herbal compounds, against AChE, was performed using Autodock 4.2 as per the standard protocols. Molecular dynamics simulations have also been carried out to check stability of binding complex in water for 1000 ps. Our molecular docking and simulation have predicted high binding affinity of secondary metabolite () to AChE. Further, molecular dynamics simulations for 1000 ps suggest that ligand interaction with the residues Asp72, Tyr70-121-334, and Phe288 of AChE, all of which fall under active site/subsite or binding pocket, might be critical for the inhibitory activity of AChE. This approach might be helpful to understand the selectivity of the given drug molecule in the treatment of Alzheimer's disease. The study provides evidence for consideration of as a valuable small ligand molecule in treatment and prevention of AD associated disorders and further in vitro and in vivo investigations may prove its therapeutic potential

A flexible low-cost quantitative phase imaging microscopy system for label-free imaging of multi-cellular biological samples

In this thesis, a flexible low-cost quantitative phase imaging microscopy (LQPIM) system for imaging both thin and thick biological phase objects in a non-contact, non-invasive, and label-free manner is reported. LQPIM optics was developed based on classical Zernike’s phase contrast approach and an additional phase shifting module to introduce user-defined phase modulations by utilising standard optical components. The phase shifting was performed using twin concentric mirrors or laser cut apertures in the arms of a Michelson interferometer where the reference mirror can be moved in / n steps (n - number of steps) with a piezoelectric transducer. Hence, the optical phase shifting modules are 10 - 15% (approximately) of the cost compared to the more widely reported modules based on spatial light modulator. In the microscope implementation reported in this thesis, a total magnification of 25x was achieved utilising relay lenses in LQPIM optics together with a standard 10x objective lens. The imaging system was simulated in MATLAB, where two-beam interference equation with varying bandwidth (1 – 250 nm), centre wavelength (450 – 650 nm) of the illumination sources and a range of previously reported phase shift algorithms (PSA) were used. The simulation results confirm that the optimum phase resolution is achievable if a broadband source of bandwidth 30 - 50 nm is used for illuminating thin (i.e. ≤ 250 nm) and thick (i.e. ≥ 1250 nm) biological samples. The four frames at 90 PSA and six plus one frames at 60 PSA offer different compromises between image acquisition time, phase resolution and out-perform other PSAs. A phase resolution of 0.382 nm and 0.317 nm was achieved using four frames at 90 and six plus one frames at 60 PSAs, respectively for the broadband illumination from a green LED. A coherent, single longitudinal mode laser source with a rotating diffuser for speckle averaging, gave 0.667 nm and 0.512 nm phase resolution using the same algorithms mentioned above. The parasitic fringes resulted in reduced resolution; hence, incoherent LED illumination was preferred. Measurements are presented over a longer optical path difference (≥ 1250 nm) than hitherto reported for a similar microscope. The given exemplar data demonstrates an ability of LQPIM system to quantify cellular and sub-cellular structures at the nanoscale in epidermis cells of Allium cepa. Key words: Quantitative phase imaging, low-cost, optical microscopy, phase imaging and phase shift imaging

In-silico studies show potent inhibition of HIV-1 Reverse Transcriptase activity by a herbal drug

Acquired immunodeficiency syndrome (AIDS) is a life threatening disease of the human immune system caused by human immunodeficiency virus (HIV). Effective inhibition of reverse transcriptase activity is a prominent, clinically viable approach for the treatment of AIDS. Few non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been approved by the United States Food and Drug Administration (US FDA) as drugs for AIDS. In order to enhance therapeutic options against AIDS we examined novel herbal compounds of 4-thiazolidinone and its derivatives that are known to have remarkable antiviral potency. Our molecular docking and simulation experiments have identified high binding affinity ligands to HIV-1RT receptor, and their consequent non-competitive inhibition binding. Results are also compared with other US FDA approved drugs. Long de novo simulations and docking study suggest that the ligand (5E)-3-(2-aminoethyl)-5-benzylidene-1, 3-thiazolidine-2,4-dione (CID: 1656714) has strong binding interactions with Asp113, Asp110, Asp185 and Asp186 amino acids, all of which belong to one or the other catalytic pockets of HIV-1RT. It is expected that these interactions could be critical in the inhibitory activity of the HIV-1RT. Therefore, this study provides an evidence for consideration of (5E)-3-(2-aminoethyl)-5-benzylidene-1, 3-thiazolidine-2,4-dione as a valuable natural molecule in the treatment and prevention of HIV- associated disorders with low toxicity to the normal cells

Role of diosgenin extracted from Helicteres isora L in suppression of HIV-1 replication: An in vitro preclinical study

Background: Diosgenin, an essential sapogenin steroid with significant biological implications, is composed of a hydrophilic sugar moiety intricately linked to a hydrophobic steroid aglycone. While the antiviral properties of diosgenin against numerous RNA viruses have been extensively documented, its potential in combating Human Immunodeficiency Virus infections remains unexplored. Experimental procedure: This current investigation presents a comprehensive and systematic analysis of extracts derived from the leaves of Helicteres isora, which are notably enriched with diosgenin. Rigorous methodologies, including established chromatographic techniques and Fourier-transform infrared spectroscopy were employed for the characterization of the active diosgenin compound followed by molecular interaction analyses with the key HIV enzymes and mechanistic validation of HIV inhibition. Key results: The inhibitory effects of extracted diosgenin on the replication of HIV-1 were demonstrated using a permissive cellular system, encompassing two distinct subtypes of HIV-1 strains. Computational analyses involving molecular interactions highlighted the substantial occupancy of critical active site pocket residues within the key HIV-1 proteins by diosgenin. Additionally, the mechanistic underpinnings of diosgenin activity in conjunction with standard controls were elucidated through specialized colorimetric assays, evaluating its impact on HIV-1 Reverse Transcriptase and Integrase enzymes. Conclusions: To our current state of knowledge, this study represents the inaugural demonstration of the anti-HIV efficacy inherent to diosgenin found in the leaves of Helicteres isora, and can be taken further for drug design and development for the management of HIV infection

Elucidation of Antiviral and Antioxidant Potential of C-Phycocyanin against HIV-1 Infection through In Silico and In Vitro Approaches

Antiretroviral therapy is the single existing therapy for patients infected with HIV; however, it has drawbacks in terms of toxicity and resistance. Thus, there is a continuous need to explore safe and efficacious anti-retroviral agents. C-Phycocyanin (C-PC) is a phycobiliprotein, which has been known for various biological properties; however, its effect on HIV-1 replication needs revelation. This study aimed to identify the inhibitory effects of C-PC on HIV-1 using in vitro and in silico approaches and to assess its role in the generation of mitochondrial reactive oxygen species (ROS) during HIV-1 infection. In vitro anti-HIV-1 activity of C-PC was assessed on TZM-bl cells through luciferase gene assay against four different clades of HIV-1 strains in a dose-dependent manner. Results were confirmed in PBMCs, using the HIV-1 p24 antigen assay. Strong associations between C-PC and HIV-1 proteins were observed through in silico molecular simulation-based interactions, and the in vitro mechanistic study confirmed its target by inhibition of reverse transcriptase and protease enzymes. Additionally, the generation of mitochondrial ROS was detected by the MitoSOX and DCF-DA probe through confocal microscopy. Furthermore, our results confirmed that C-PC treatment notably subdued the fluorescence in the presence of the virus, thus reduction of ROS and the activation of caspase-3/7 in HIV-1-infected cells. Overall, our study suggests C-PC as a potent and broad in vitro antiviral and antioxidant agent against HIV-1 infection

Withania somnifera extracts induced attenuation of HIV-1: a mechanistic approach to restrict viral infection

Abstract Background Several anti-retroviral drugs are available against Human immunodeficiency virus type-1, but have multiple adverse side effects. Hence, there is an incessant compulsion for effectual anti-retroviral agents with minimal or no intricacy. Traditionally, natural products have been the most successful source for the development of new medications. Withania somnifera, also known as Ashwagandha, is the utmost treasured medicinal plant used in Ayurveda, which holds the potential to give adaptogenic, immunomodulatory, and antiviral effects. However, its effect on HIV-1 replication at the cellular level has never been explored. Herein, we focused on the anti-HIV-1 activity and the probable mechanism of action of hydroalcoholic and aqueous extracts of Withania somnifera roots and its phytomolecules. Methods The cytotoxicity of the extracts was determined through MTT assay, while the in vitro anti-HIV-1 activity was assessed in TZM-bl cells against the HIV-1 strains of X4 and R5 subtypes. Results were confirmed in peripheral blood mononuclear cells, using the HIV-1 p24 antigen assay. Additionally, the mechanism of action was determined through the Time of Addition assay, which was further validated through the series of enzymatic assays, i.e. HIV-1 Integrase, Reverse transcriptase, and Protease assays. To explore the role of the identified active metabolites of Withania somnifera in antiretroviral activity, molecular docking analyses were performed against these key HIV-1 replication enzymes. Results The hydroalcoholic and aqueous extracts of Withania somnifera roots were found to be safer at the sub-cytotoxic concentrations and exhibited their ability to inhibit replication of two primary isolates of HIV-1 through cell-associated and cell-free assays, in dose-dependent kinetics. Several active phytomolecules found in Withania somnifera successfully established hydrogens bonds in the active binding pocket site residues responsible for the catalytic activity of HIV replication and therefore, signifying their role in the attenuation of HIV-1 infection as implied through the in silico molecular docking studies. Conclusions Our research identified both the hydroalcoholic and aqueous extracts of Withania somnifera roots as potent inhibitors of HIV-1 infection. The in silico analyses also indicated the key components of Withania somnifera with the highest binding affinity against the HIV-1 Integrase by 12-Deoxywithastramonolide and 27-Hydroxywithanone, HIV-1 Protease by Ashwagandhanolide and Withacoagin, and HIV-1 Reverse transcriptase by Ashwagandhanolide and Withanolide B, thereby showing possible mechanisms of HIV-1 extenuation. Overall, this study classified the role of Withania somnifera extracts and their active compounds as potential agents against HIV-1 infection