Biology subjects

Perera, T.

Publications and source records attributed to Perera, T..

11 recordsLinked to original sources

bioRxiv2024

First report of pathogenic Leptospira in Sri Lankan bats: A potential reservoir risk

BackgroundLeptospirosis is a bacterial infection caused by spirochetes of the genus Leptospira. It affects both humans and animals, posing a significant public health threat worldwide. In Sri Lanka, leptospirosis causes annual outbreaks, particularly during the monsoon seasons. The disease is often difficult to diagnose and can have a high mortality rate. Bats are potential reservoirs for Leptospira in other countries. This study investigated the presence and diversity of Leptospira in bats inhabiting the Wavulgalge, a natural cave in Sri Lanka. Methodology/Principal FindingsUsing PCR and Sanger sequencing techniques, Leptospira borgpetersenii and Leptospira kirschneri were identified in Miniopterus fuliginosus and Rousettus leschenaultii bats. Out of 148 bat urine samples, 16 tested positive via RT-qPCR, with M. fuliginosus samples harboring L. borgpetersenii and one R. leschenaultii sample harboring L. kirschneri revealed by Sanger sequencing method. Conclusions/SignificanceThese findings enhance the understanding of the zoonotic role of Sri Lankan bats in Leptospira transmission. Author SummaryLeptospirosis is a bacterial disease that affects both humans and animals, particularly in tropical regions. It spreads through contact with water or soil contaminated by the urine of infected animals. In Sri Lanka, leptospirosis poses a significant public health challenge, with frequent outbreaks during the rainy season. However, while much research has focused on domestic animals as potential carriers, the role of bats in the transmission of this disease has not been fully explored. In this study, we investigated whether bats in Sri Lanka can act as carriers of Leptospira bacteria, which could contribute to the spread of leptospirosis to humans. We focused on a population of bats from the Wavulgalge cave and tested them for the presence of Leptospira using molecular methods. Our results provide the first evidence that Leptospira is present in Sri Lankan bats, highlighting their potential role as a reservoir for the bacteria. These findings are important because they suggest that bats, like rodents and livestock, could contribute to the spread of leptospirosis in Sri Lanka. Understanding which animals carry the bacteria helps inform public health strategies, allowing for more targeted surveillance and control measures. By including bats in these efforts, we can better prevent future outbreaks of leptospirosis and protect at-risk communities. This study also contributes to the broader understanding of how wildlife can influence human health, especially in tropical environments where human-animal interactions are common. Our work emphasizes the need for a One Health approach, which considers the connections between human, animal, and environmental health, to tackle neglected diseases like leptospirosis.

microbiology↗

bioRxiv2024

First Report of Nipah virus shed in urine by fruit bats (Pteropus medius), Sri Lanka

Nipah virus is an emerging pathogen with high public health relevance, causing outbreaks in South- and Southeast Asia with high case fatality rates. The natural reservoir host are fruit bats (Pteropus spp). In this study, we confirmed the presence of Nipah virus for the first time in Sri Lanka and are proving the genetic relatedness to Nipah virus strains from southern India, causing outbreaks in 2007, 2018, 2019 and 2023. It is noteworthy that the shedding of the Nipah Virus may have a temporal variation that correlates with the breeding season of Pteropus medius. Furthermore, preventive measures and possible mechanisms influencing viral shedding are discussed.

microbiology↗

bioRxiv2024

Quantitative Superresolution Imaging of F-Actin in the Cell Body and Cytoskeletal Protrusions Using Phalloidin-Based Single-Molecule Labeling and Localization Microscopy

We present single-molecule labeling and localization microscopy (SMLLM) using dye-conjugated phalloidin to achieve enhanced superresolution imaging of filamentous actin (F-actin). We demonstrate that the intrinsic phalloidin dissociation enables SMLLM in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-based SMLLM are better preservation of cellular structures sensitive to mechanical and shear forces during standard sample preparation and more consistent F-actin quantification at the nanoscale. In a proof-of-concept study, we employed SMLLM to super-resolve F-actin structures in U2OS and dendritic cells (DCs) and demonstrate more consistent F-actin quantification in the cell body and structurally delicate cytoskeletal proportions, which we termed membrane fibers, of DCs compared to direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse dendritic cells as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane fibers on the culture glass surface after lipopolysaccharide exposure. While our work demonstrates SMLLM for F-actin, the concept opens new possibilities for protein-specific single-molecule labeling and localization in the same step using commercially available reagents.

cell biology↗

bioRxiv2024

Probing drug-target engagement of soluble Gαi1 protein using the SolThermoBRET thermal shift assay

IntroductionSensitive protein stability assays used during hit confirmation conventionally require high quantities of purified protein. Here, we describe a novel high-throughput 384-well BRET-based thermostability assay allowing for the ultrasensitive determination of Gi1 protein stability. Using this method we make use of the environmentally sensitive dye, SYPRO Red, which interacts with hydrophobic regions in the protein that become exposed upon denaturation. Assays are functional in crude cell lysates, without any requirement for protein purification. MethodsThe SolThermoBRET method measures resonance energy transfer between a thermally stabilised nano-luciferase (tsNLuc) genetically engineered to the N-terminus of Gi1 protein, and SYPRO Red, a fluorescent dye that binds to lipophilic residues exposed upon protein unfolding in response to thermal denaturation. Samples in 384-well PCR plate were subjected to a temperature gradient (30 - 60 {degrees}C) on a PCR block and incubated for 5 minutes. Isothermal SolThermoBRET at a fixed temperature 45{degrees}C allowed the measurement of IC50 curves to various ligands. Furimazine (10 M) was added to the samples and BRET read on the BMG Labtech PHERAstar FSX at room temperature. Melt curves were fit to a Boltzmann sigmoidal equation to obtain Tm values. ResultsLigand-induced stabilisation of Gi1 protein was demonstrated in non-purified samples. A range of Tm values were measured with increasing stabilisation observed for nucleotides/sides in the following order GDP<GTP<GppNHp<GTP{gamma}S. Correlation was observed between SolThermoBRET derived Tms determined in the presence of high ligand concentrations and IC50 determinations using Isothermal SolThermoBRET. ConclusionsSolThermoBRET represents a sensitive nanoscale method for the detection of changes in soluble protein thermostability. This method is attractive for hit conformation and the identification of novel ligands targeting soluble proteins.

biophysics↗

bioRxiv2022

One Health Approach for the sampling of different bat species living in a sympatric colony

Bats are important contributors to the global ecosystems; at the same time, they are known to be a natural reservoir host for a number of human pathogenic viruses. These and many other unique features make them an interdisciplinary research object in the context of One Health, comprising zoology, ecology, virology, microbiology, molecular biology, immunology and public health issues. Performing field studies for bat research often aims to cover several of these topics and requires the combination of specific expertise in different fields. We carried out three individual field studies in Wavul Galge cave (Koslanda, Sri Lanka), where several bat species roost sympatrically. The main goals were to study the bat colony for ecological aspects and to sample bats for virological and molecular biological analyses. In the course of the field studies, we optimized the sampling procedure regarding safety aspects, a preferably low impact on the captured bats and an improved output of high-quality samples for further analysis. Different sampling methods and procedures were compared in order to establish a suitable strategy for frequent sampling and monitoring of these bats. In the present case study, we report on this process of optimizing our field work and provide suggestions for bat sampling methods that cause comparably less stress for the captured animals. We also report on constraints and obstacles encountered during the practical implementation and possible measures to overcome these. With these practical experiences, we hope to give support to other interdisciplinary research teams preparing for bat field work. Furthermore, we emphasize the need for the respectful treatment of the animals and minimized disturbance of their natural habitat when carrying out sustainable bat research.

zoology↗

bioRxiv2022

Comparative virome analysis of individual shedding routes of Miniopterus fuliginosus bats inhabiting the Wavul Galge Cave, Sri Lanka

Bats are described as the natural reservoir host for a wide range of viruses. Although an increasing number of bat-associated, potentially human pathogenic viruses were discovered in the past, the full picture of the bat viromes is not explored yet. In this study, the virome composition from Miniopterus fuliginosus bats inhabiting the Wavul Galge cave, Sri Lanka, was analyzed. To assess different possible shedding routes, oral swabs, feces and urine were collected and analyzed individually by using metagenomic NGS. The data obtained was further evaluated by using phylogenetic reconstructions. Two different alphacoronavirus strains were detected in feces and urine samples. Furthermore, a paramyxovirus was detected in urine samples. Sequences related to Picornaviridae, Iflaviridae, unclassified Riboviria and Astroviridae were identified in feces samples, and further sequences related to Astroviridae in urine samples. No further viruses were detected in oral swab samples. The comparative virome analysis in this study revealed a diversity in the virome composition between the collected sample types which also represent different potential shedding routes for the detected viruses. At the same time, several viruses were detected for the first time in bats in Sri Lanka. The detection of two different coronaviruses in the samples indicates the potential general persistence of this virus species in M. fuliginosus bats. Based on phylogenetics, the identified viruses are closer related to bat-associated viruses with comparably low human pathogenic potential. In further studies, the seasonal variation of the virome will be analyzed to identify possible shedding patterns for particular viruses.

molecular biology↗

bioRxiv2022

Sudden unexpected death in epilepsy is prevented by blocking postictal hypoxia

Epilepsy is at times a fatal disease. Sudden unexpected death in epilepsy (SUDEP) is the leading cause of mortality in people with intractable epilepsy and is defined by exclusion; non-accidental, non-toxicologic, and non-anatomic causes of death. While SUDEP often follows a bilateral tonic-clonic seizure, the mechanisms that ultimately lead to terminal apnea and then asystole remain elusive and there is a lack preventative treatments. Based on the observation that discrete seizures lead to local vasoconstriction, resulting in hypoperfusion, hypoxia and behavioural disturbances in the forebrain (Farrell et al., 2016), we reasoned that similar mechanisms may play a role in SUDEP when seizures invade the brainstem. Here we tested this neurovascular-based hypothesis of SUDEP in awake non-anesthetized mice by pharmacologically preventing seizure-induced vasoconstriction, with cyclooxygenase-2 or L-type calcium channel antagonists. In both acute and chronic mouse models of SUDEP, ibuprofen and nicardipine extended life. We also examined the potential role of spreading depolarization in the acute model of SUDEP. These data provide a proof of principle for the neurovascular hypothesis of SUDEP and the use of currently available treatments to prevent it.

neuroscience↗

bioRxiv2021

Molecular identification of bats from Wavulgalge cave, Wellawaya, Sri Lanka

This is the first report on the molecular identification and phylogeny of Rousettus leschenaultii, Rhinolophus rouxii, Hipposideros speoris, Hipposideros lankadiva, Miniopterus fuliginosus bat species in Sri Lanka, inferred from mitochondrially encoded cytochrome b gene sequences. Wellawaya Wavulgalge cave in Sri Lanka is one of the largest sympatric colonies found on the island, occupied by five species of bats. Recent research has indicated that bats show enormous cryptic genetic diversity. Moreover, even in the same species, acoustic properties of echolocation calls and morphological features such as fur colour could vary in different populations. Therefore, we have used molecular techniques for the accurate identification of five bat species recorded in one of the largest cave populations in Sri Lanka. Bats were caught using a hand net and saliva samples were collected non-invasively from each bat using a sterile oral swab. Nucleic acids were extracted from oral swab samples and mitochondrial DNA was amplified using primers targeting the mitochondrially encoded cytochrome b gene. This study identified the bat species recorded in the Wellawaya cave as Rousettus leschenaultii, Rhinolophus rouxii, Hipposideros speoris, Hipposideros lankadiva and Miniopterus fuliginosus. Our findings will contribute to future conservation and systematic studies of bats in Sri Lanka. This study will also provide the basis for a genetic database of Sri Lankan bats.

zoology↗

bioRxiv2020

Single-molecule interaction microscopy reveals antibody binding kinetics

Single-molecule imaging has provided new insights on weak transient biomolecular interactions with micromolar to millimolar affinity. However, the limited duration of observation has hindered the study of strong and reversible interactions with sub-nanomolar affinity. We report single-molecule interaction microscopy (SMIM), which combines point accumulation for imaging in nanoscale topography (PAINT) with extended imaging durations that enables the study of antibody binding kinetics in the cellular environment. SMIM revealed heterogeneous binding kinetics and the effect of concentration and antibody valency on the association and dissociation rates on antibody-antigen interactions in their cellular environments. We thereby demonstrate SMIM as a versatile single-molecule technique for studying strong, transient biomolecular interactions.

biophysics↗

bioRxiv2020

Evaluation of cerebellar function scores in relation to cerebellar axonal loss in multiple sclerosis

BackgroundCerebellar damage is common in people with multiple sclerosis (pwMS) and is associated with worse progression and relapse recovery. Studies into the importance of the cerebellum in pwMS are hampered by limited understanding of cerebellar damage and its relation to cerebellar function in pwMS. ObjectiveExamine axonal loss, as a primary driver of progressive neurological decline, in the cerebellum using advanced diffusion MRI and compare axonal loss with cerebellar dysfunction in pwMS MethodsWe recruited 55 pwMS and 14 healthy controls. Clinical assessments included scale for the assessment and rating of ataxia (SARA), and Bain tremor ratings. Subjects underwent FLAIR, T1-weighted and diffusion MRI. Cerebellar grey and white matter and lesion volume were calculated. Cerebellar axonal loss was examined with fibre-specific markers. Fibre density and cross-section (FDC) accounts for microscopic and macroscopic changes in a fibre bundle. ResultsLoss of cerebellar FDC was associated with increased SARA (r=-0.42, p<0.01) and tremor severity (rho=-0.35, p=0.01). Cerebellar lesion volume correlated with SARA (r=0.49, p<0.01) and tremor severity (rho=0.41, p=0.01). ConclusionFibre-specific measures of cerebellar pathology could provide a functionally relevant marker of cerebellar damage in MS. Future trials using fibre-specific markers are needed to further characterize cerebellar pathology in pwMS and understand its significance in disease progression.

neuroscience↗

bioRxiv2018

Lead-DBS v2: Toward a comprehensive pipeline for deep brain stimulation imaging

Deep brain stimulation (DBS) is a highly efficacious treatment option for movement disorders and a growing number of other indications are investigated in clinical trials. To ensure optimal treatment outcome, exact electrode placement is required. Moreover, to analyze the relationship between electrode location and clinical results, a precise reconstruction of electrode placement is required, posing specific challenges to the field of neuroimaging. Since 2014 the open source toolbox Lead-DBS is available, which aims at facilitating this process. The tool has since become a popular platform for DBS imaging. With support of a broad community of researchers worldwide, methods have been continuously updated and complemented by new tools for tasks such as multispectral nonlinear registration, structural / functional connectivity analyses, brain shift correction, reconstruction of microelectrode recordings and orientation detection of segmented DBS leads. The rapid development and emergence of these methods in DBS data analysis require us to revisit and revise the pipelines introduced in the original methods publication. Here we demonstrate the updated DBS and connectome pipelines of Lead-DBS using a single patient example with state-of-the-art high-field imaging as well as a retrospective cohort of patients scanned in a typical clinical setting at 1.5T. Imaging data of the 3T example patient is co-registered using five algorithms and nonlinearly warped into template space using ten approaches for comparative purposes. After reconstruction of DBS electrodes (which is possible using three methods and a specific refinement tool), the volume of tissue activated is calculated for two DBS settings using four distinct models and various parameters. Finally, four whole-brain tractography algorithms are applied to the patients preoperative diffusion MRI data and structural as well as functional connectivity between the stimulation volume and other brain areas are estimated using a total of eight approaches and datasets. In addition, we demonstrate impact of selected preprocessing strategies on the retrospective sample of 51 PD patients. We compare the amount of variance in clinical improvement that can be explained by the computer model depending on the method of choice.\n\nThis work represents a multi-institutional collaborative effort to develop a comprehensive, open source pipeline for DBS imaging and connectomics, which has already empowered several studies, and may facilitate a variety of future studies in the field.

neuroscience↗