Search results for: Bidya Dhar Sahu

Authors: Umesh Regmi

Abstract:

This paper explores the expansion of Buddhism from India geographically to the Himalayan region of Nepal, Tibet, India, and Bhutan in chronological historical sequence. The Buddhism practiced in Tibet is the spread of the Mahayana-Vajrayana form appropriately designed by Indian Mahasiddhas, who were the practitioners of the highest form of tantra and meditation. Vajrayana Buddhism roots in the esoteric practices incorporating the teachings of Buddha, mantras, dharanis, rituals, and sadhana for attaining enlightenment. This form of Buddhism spread from India to Nepal after the 5th Century AD and Tibet after the 7th century AD and made a return journey to the Himalayan region of Nepal, India, and Bhutan after the 8th century. The first diffusion of this form of Buddhism from India to Nepal and Tibet is partially proven through Buddhist texts and the archaeological existence of monasteries historically and at times relied on mythological traditions. The second diffusion of Buddhism in Tibet was institutionalized through the textual translations and interpretations of Indian Buddhist masters and their Tibetan disciples and the establishment of different monasteries in various parts of Tibet, later resulting in different schools and their traditions: Nyingma, Kagyu, Sakya, Gelug, and their sub-schools. The first return journey of Buddhism from Tibet to the Himalayan region of Nepal, India, and Bhutan in the 8th century is mythologically recorded in local legends of the arrival of Padmasambhava, and the second journey in the 11th century and afterward flourished by many Indian masters who practiced continuously till date. This return journey of Tibetan Buddhism has been intensified after 1959 with the Chinese occupation of Tibet, resulting in the Tibetan Buddhist masters living in exile in major locations like Kathmandu, Dharmasala, Dehradun, Sikkim, Kalimpong, and beyond. The historic-cultural-critical methodology for the recognition of the qualities of cultural expressions analysis presents the Buddhist practices of the Himalayan region, explaining the concepts of Ri (mountain as spiritual symbols), yul-lha (village deities), dhar-lha (spiritual concept of mountain passes), dharchhog-lungdhar (prayer flags), rig-sum gonpo (small stupas), Chenresig, asura (demi gods), etc. Tibetan Buddhist history has preserved important textual and practical aspects of Vajrayana from Buddhism historically in the form of arrival, advent, and development, including rising and fall. Currently, Tibetan Buddhism has influenced a great deal in the contemporary Buddhist practices of the world. The exploratory findings conducted over seven years of field visits and research in the Himalayan regions of Nepal, India, and Bhutan have demonstrated the fact that Buddhism in the Himalayan region is a return journey from Tibet and lately been popularized globally after 1959 by major monasteries and their Buddhist masters, lamas, nuns and other professionals, who have contributed in different periods of time.

Keywords: Buddhism, expansion, Himalayan region, India, Nepal, Bhutan, return, Tibet, Vajrayana Buddhism

Procedia PDF Downloads 73

Authors: Tapan Kumar Dhar

Abstract:

How well are our city forms designed to adapt to climate change and its resulting uncertainty? What urban design tools can be used to measure and improve resilience to climate change, and how would they do so? In addressing these questions, this paper considers indeterminacy, a concept originated in the resilience literature, to measure the resilience of built environments. In the realm of urban design, ‘indeterminacy’ can be referred to as built-in design capabilities of an urban system to serve different purposes which are not necessarily predetermined. An urban system, particularly that with a higher degree of indeterminacy, can enable the system to be reorganized and changed to accommodate new or unknown functions while coping with uncertainty over time. Underlying principles of this concept have long been discussed in the urban design and planning literature, including open architecture, landscape urbanism, and flexible housing. This paper argues that the concept indeterminacy holds the potential to reduce the impacts of climate change incrementally and proactively. With regard to sustainable development, both planning and climate change literature highly recommend proactive adaptation as it involves less cost, efforts, and energy than last-minute emergency or reactive actions. Nevertheless, the concept still remains isolated from resilience and climate change adaptation discourses even though the discourses advocate the incremental transformation of a system to cope with climatic uncertainty. This paper considers indeterminacy, as an urban design tool, to measure and increase resilience (and adaptive capacity) of Long Bay’s coastal settlements in Negril, Jamaica. Negril is one of the popular tourism destinations in the Caribbean highly vulnerable to sea-level rise and its associated impacts. This paper employs empirical information obtained from direct observation and informal interviews with local people. While testing the tool, this paper deploys an urban morphology study, which includes land use patterns and the physical characteristics of urban form, including street networks, block patterns, and building footprints. The results reveal that most resorts in Long Bay are designed for pre-determined purposes and offer a little potential to use differently if needed. Additionally, Negril’s street networks are found to be rigid and have limited accessibility to different points of interest. This rigidity can expose the entire infrastructure further to extreme climatic events and also impedes recovery actions after a disaster. However, Long Bay still has room for future resilient developments in other relatively less vulnerable areas. In adapting to climate change, indeterminacy can be reached through design that achieves a balance between the degree of vulnerability and the degree of indeterminacy: the more vulnerable a place is, the more indeterminacy is useful. This paper concludes with a set of urban design typologies to increase the resilience of coastal settlements.

Keywords: climate change adaptation, resilience, sea-level rise, urban form

Procedia PDF Downloads 331

Authors: Turin Datta, Kisor K. Sahu

Abstract:

Structural materials used in today’s oil and gas exploration and drilling of both onshore and offshore oil and gas wells must possess superior tensile properties, excellent resistance to corrosive degradation that includes general, localized (pitting and crevice) and environment assisted cracking such as stress corrosion cracking and hydrogen embrittlement. The High Pressure and High Temperature (HPHT) wells are typically operated at temperature and pressure that can exceed 300-3500F and 10,000psi (69MPa) respectively which necessitates the use of exotic materials in these exotic sources of natural resources. This research investigation is focussed on the evaluation of tensile properties and corrosion behavior of AISI 4140 High-Strength Low Alloy Steel (HSLA) possessing tempered martensitic microstructure and Duplex 2205 Stainless Steel (DSS) having austenitic and ferritic phase. The selection of this two alloys are primarily based on economic considerations as 4140 HSLA is cheaper when compared to DSS 2205. Due to the harsh aggressive chemical species encountered in deep oil and gas wells like chloride ions (Cl-), carbon dioxide (CO2), hydrogen sulphide (H2S) along with other mineral organic acids, DSS 2205, having a dual-phase microstructure can mitigate the degradation resulting from the presence of both chloride ions (Cl-) and hydrogen simultaneously. Tensile properties evaluation indicates a ductile failure of DSS 2205 whereas 4140 HSLA exhibit quasi-cleavage fracture due to the phenomenon of ‘tempered martensitic embrittlement’. From the potentiodynamic polarization testing, it is observed that DSS 2205 has higher corrosion resistance than 4140 HSLA; the former exhibits passivity signifying resistance to localized corrosion while the latter exhibits active dissolution in all the environmental parameters space that was tested. From the Scanning Electron Microscopy (SEM) evaluation, it is understood that stable pits appear in DSS 2205 only when the temperature exceeds the critical pitting temperature (CPT). SEM observation of the corroded 4140 HSLA specimen tested in aqueous 3.5 wt.% NaCl solution reveals intergranular cracking which appears due to the adsorption and diffusion of hydrogen during polarization, thus, causing hydrogen-induced cracking/hydrogen embrittlement. General corrosion testing of DSS 2205 in acidic brine (pH~3.0) solution at ambient temperature using coupons indicate no weight loss even after three months whereas the corrosion rate of AISI 4140 HSLA is significantly higher after one month of testing.

Keywords: DSS 2205, polarization, pitting, SEM

Procedia PDF Downloads 238

Authors: Neha Sahu, Awantika Singh, Brijesh Kumar, K. R. Arya

Abstract:

Taraxacum officinale Weber or dandelion (Asteraceae) is an important Indian traditional herb used to treat liver detoxification, digestive problems, spleen, hepatic and kidney disorders, etc. The plant is well known to possess important phenolic and flavonoids to serve as a potential source of antioxidative and chemoprotective agents. Biosynthesis of bioactive compounds through in vitro cultures is a requisite for natural resource conservation and to provide an alternative source for pharmaceutical applications. Thus an efficient and reproducible protocol was developed for in vitro biosynthesis of bioactive antioxidative compounds from leaf derived callus and in vitro regenerated cultures of Taraxacum officinale using MS media fortified with various combinations of auxins and cytokinins. MS media containing 0.25 mg/l 2, 4-D (2, 4-Dichloro phenoxyacetic acid) with 0.05 mg/l 2-iP [N6-(2-Isopentenyl adenine)] was found as an effective combination for the establishment of callus with 92 % callus induction frequency. Moreover, 2.5 mg/l NAA (α-Naphthalene acetic acid) with 0.5 mg/l BAP (6-Benzyl aminopurine) and 1.5 mg/l NAA showed the optimal response for in vitro plant regeneration with 80 % regeneration frequency and rooting respectively. In vitro regenerated plantlets were further transferred to soil and acclimatized. Quantitative variability of accumulated bioactive compounds in cultures (in vitro callus, plantlets and acclimatized) were determined through UPLC-MS/MS (ultra-performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry) and compared with wild plants. The phytochemical determination of in vitro and wild grown samples showed the accumulation of 6 compounds. In in vitro callus cultures and regenerated plantlets, two major antioxidative compounds i.e. chlorogenic acid (14950.0 µg/g and 4086.67 µg/g) and umbelliferone (10400.00 µg/g and 2541.67 µg/g) were found respectively. Scopoletin was found to be highest in vitro regenerated plants (83.11 µg/g) as compared to wild plants (52.75 µg/g). Notably, scopoletin is not detected in callus and acclimatized plants, but quinic acid (6433.33 µg/g) and protocatechuic acid (92.33 µg/g) were accumulated at the highest level in acclimatized plants as compared to other samples. Wild grown plants contained highest content (948.33 µg/g) of flavonoid glycoside i.e. luteolin-7-O-glucoside. Our data suggests that in vitro callus and regenerated plants biosynthesized higher content of antioxidative compounds in controlled conditions when compared to wild grown plants. These standardized cultural conditions may be explored as a sustainable source of plant materials for enhanced production and adequate supply of oxidative polyphenols.

Keywords: anti-oxidative compounds, in vitro cultures, Taraxacum officinale, UPLC-MS/MS

Procedia PDF Downloads 177

Authors: Ahmad Saadiq, Neeraj Sahu

Abstract:

Sediment, which comprises of solid particles of mineral and organic material are transported by water. In river systems, the amount of sediment transported is controlled by both the transport capacity of the flow and the supply of sediment. The transport of sediment in rivers is important with respect to pollution, channel navigability, reservoir ageing, hydroelectric equipment longevity, fish habitat, river aesthetics and scientific interests. The sediment load transported in a river is a very complex hydrological phenomenon. Hence, sediment transport has attracted the attention of engineers from various aspects, and different methods have been used for its estimation. So, several experimental equations have been submitted by experts. Though the results of these methods have considerable differences with each other and with experimental observations, because the sediment measures have some limits, these equations can be used in estimating sediment load. In this present study, two black box models namely, an SRC (Sediment Rating Curve) and ANN (Artificial Neural Network) are used in the simulation of the suspended sediment load. The study is carried out for Seonath subbasin. Seonath is the biggest tributary of Mahanadi river, and it carries a vast amount of sediment. The data is collected for Jondhra hydrological observation station from India-WRIS (Water Resources Information System) and IMD (Indian Meteorological Department). These data include the discharge, sediment concentration and rainfall for 10 years. In this study, sediment load is estimated from the input parameters (discharge, rainfall, and past sediment) in various combination of simulations. A sediment rating curve used the water discharge to estimate the sediment concentration. This estimated sediment concentration is converted to sediment load. Likewise, for the application of these data in ANN, they are normalised first and then fed in various combinations to yield the sediment load. RMSE (root mean square error) and R² (coefficient of determination) between the observed load and the estimated load are used as evaluating criteria. For an ideal model, RMSE is zero and R² is 1. However, as the models used in this study are black box models, they don’t carry the exact representation of the factors which causes sedimentation. Hence, a model which gives the lowest RMSE and highest R² is the best model in this study. The lowest values of RMSE (based on normalised data) for sediment rating curve, feed forward back propagation, cascade forward back propagation and neural network fitting are 0.043425, 0.00679781, 0.0050089 and 0.0043727 respectively. The corresponding values of R² are 0.8258, 0.9941, 0.9968 and 0.9976. This implies that a neural network fitting model is superior to the other models used in this study. However, a drawback of neural network fitting is that it produces few negative estimates, which is not at all tolerable in the field of estimation of sediment load, and hence this model can’t be crowned as the best model among others, based on this study. A cascade forward back propagation produces results much closer to a neural network model and hence this model is the best model based on the present study.

Keywords: artificial neural network, Root mean squared error, sediment, sediment rating curve

Procedia PDF Downloads 297

Authors: Jonathan Sahu, Jill Aylott

Abstract:

Individuals with Autism, Intellectual and Developmental disabilities (AIDD) are one of the most vulnerable groups in society, hampered not only by their own limitations to understand and interact with the wider society, but also societal limitations in perception and understanding. Communication to express their needs and wishes is fundamental to enable such individuals to live and prosper in society. This research project was designed as an organisational case study, in a large secondary health care hospital within the National Health Service (NHS), to assess the quality of care provided to people with AIDD and to review the role of advocacy to reduce health inequalities in these individuals. Methods: The research methodology adopted was as an “insider researcher”. Data collection included both quantitative and qualitative data i.e. a mixed method approach. A semi-structured interview schedule was designed and used to obtain qualitative and quantitative primary data from a wide range of interdisciplinary frontline health care workers to assess their understanding and awareness of systems, processes and evidence based practice to offer a quality service to people with AIDD. Secondary data were obtained from sources within the organisation, in keeping with “Case Study” as a primary method, and organisational performance data were then compared against national benchmarking standards. Further data sources were accessed to help evaluate the effectiveness of different types of advocacy that were present in the organisation. This was gauged by measures of user and carer experience in the form of retrospective survey analysis, incidents and complaints. Results: Secondary data demonstrate near compliance of the Organisation with the current national benchmarking standard (Monitor Compliance Framework). However, primary data demonstrate poor knowledge of the Mental Capacity Act 2005, poor knowledge of organisational systems, processes and evidence based practice applied for people with AIDD. In addition there was poor knowledge and awareness of frontline health care workers of advocacy and advocacy schemes for this group. Conclusions: A significant amount of work needs to be undertaken to improve the quality of care delivered to individuals with AIDD. An operational strategy promoting the widespread dissemination of information may not be the best approach to deliver quality care and optimal patient experience and patient advocacy. In addition, a more robust set of standards, with appropriate metrics, needs to be developed to assess organisational performance which will stand the test of professional and public scrutiny.

Keywords: advocacy, autism, health inequalities, intellectual developmental disabilities, quality of care

Procedia PDF Downloads 191

Authors: Jana Chakrabarti, Madhushrita Das, Ankhi Haldar, Roshni Chatterjee, Tanmoy Dey, Pubali Dhar

Abstract:

High incidences of Protein Energy Malnutrition as a consequence of low protein intake are quite prevalent among the children in developing countries. Thus prevention of under-nutrition has emerged as a critical challenge to India’s developmental Planners in recent times. Increase in population over the last decade has led to greater pressure on the existing animal protein sources. But these resources are currently declining due to persistent drought, diseases, natural disasters, high-cost of feed, and low productivity of local breeds and this decline in productivity is most evident in some developing countries. So the need of the hour is to search for efficient utilization of unconventional low-cost animal protein resources. Molluscs, as a group is regarded as under-exploited source of health-benefit molecules. Bivalve is the second largest class of phylum Mollusca. Annual harvests of bivalves for human consumption represent about 5% by weight of the total world harvest of aquatic resources. The freshwater mussel Lamellidens marginalis is widely distributed in ponds and large bodies of perennial waters in the Indian sub-continent and well accepted as food all over India. Moreover, ethno-medicinal uses of the flesh of Lamellidens among the rural people to treat hypertension have been documented. Present investigation thus attempts to evaluate the potential of Lamellidens marginalis as functional food. Mussels were collected from freshwater ponds and brought to the laboratory two days before experimentation for acclimatization in laboratory conditions. Shells were removed and fleshes were preserved at- 20oC until analysis. Tissue homogenate was prepared for proximate studies. Fatty acids and amino acids composition were analyzed. Vitamins, Minerals and Heavy metal contents were also studied. Mussel Protein hydrolysate was prepared using Alcalase 2.4 L and degree of hydrolysis was evaluated to analyze its Functional properties. Ferric Reducing Antioxidant Power (FRAP) and DPPH Antioxidant assays were performed. Anti-hypertensive property was evaluated by measuring Angiotensin Converting Enzyme (ACE) inhibition assay. Proximate analysis indicates that mussel meat contains moderate amount of protein (8.30±0.67%), carbohydrate (8.01±0.38%) and reducing sugar (4.75±0.07%), but less amount of fat (1.02±0.20%). Moisture content is quite high but ash content is very low. Phospholipid content is significantly high (19.43 %). Lipid constitutes, substantial amount of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which have proven prophylactic values. Trace elements are found present in substantial amount. Comparative study of proximate nutrients between Labeo rohita, Lamellidens and cow’s milk indicates that mussel meat can be used as complementary food source. Functionality analyses of protein hydrolysate show increase in Fat absorption, Emulsification, Foaming capacity and Protein solubility. Progressive anti-oxidant and anti-hypertensive properties have also been documented. Lamellidens marginalis can thus be regarded as a functional food source as this may combine effectively with other food components for providing essential elements to the body. Moreover, mussel protein hydrolysate provides opportunities for utilizing it in various food formulations and pharmaceuticals. The observations presented herein should be viewed as a prelude to what future holds.

Keywords: functional food, functional properties, Lamellidens marginalis, protein hydrolysate

Procedia PDF Downloads 390

Authors: Jyoti Sahu, Vinay A. Juvekar

Abstract:

Electrolytes are often used to bring about salting-in and salting-out of organic molecules and polymers (e.g. polyethylene glycols/proteins) from the aqueous solutions. For quantification of these phenomena, a thermodynamic model which can accurately predict activity coefficient of electrolyte as a function of temperature is needed. The thermodynamics models available in the literature contain a large number of empirical parameters. These parameters are estimated using lower/upper critical solution temperature of the solution in the electrolyte/organic molecule at different temperatures. Since the number of parameters is large, inaccuracy can bethe creep in during their estimation, which can affect the reliability of prediction beyond the range in which these parameters are estimated. Cloud point of solution is related to its free energy through temperature and composition derivative. Hence, the Cloud point measurement can be used for accurate estimation of the temperature and composition dependence of parameters in the model for free energy. Hence, if we use a two pronged procedure in which we first use cloud point of solution to estimate some of the parameters of the thermodynamic model and determine the rest using osmotic coefficient data, we gain on two counts. First, since the parameters, estimated in each of the two steps, are fewer, we achieve higher accuracy of estimation. The second and more important gain is that the resulting model parameters are more sensitive to temperature. This is crucial when we wish to use the model outside temperatures window within which the parameter estimation is sought. The focus of the present work is to prove this proposition. We have used electrolyte (NaCl/Na2CO3)-water-organic molecule (Iso-propanol/ethanol) as the model system. The model of Robinson-Stokes-Glukauf is modified by incorporating the temperature dependent Flory-Huggins interaction parameters. The Helmholtz free energy expression contains, in addition to electrostatic and translational entropic contributions, three Flory-Huggins pairwise interaction contributions viz., and (w-water, p-polymer, s-salt). These parameters depend both on temperature and concentrations. The concentration dependence is expressed in the form of a quadratic expression involving the volume fractions of the interacting species. The temperature dependence is expressed in the form .To obtain the temperature-dependent interaction parameters for organic molecule-water and electrolyte-water systems, Critical solution temperature of electrolyte -water-organic molecules is measured using cloud point measuring apparatus The temperature and composition dependent interaction parameters for electrolyte-water-organic molecule are estimated through measurement of cloud point of solution. The model is used to estimate critical solution temperature (CST) of electrolyte water-organic molecules solution. We have experimentally determined the critical solution temperature of different compositions of electrolyte-water-organic molecule solution and compared the results with the estimates based on our model. The two sets of values show good agreement. On the other hand when only osmotic coefficients are used for estimation of the free energy model, CST predicted using the resulting model show poor agreement with the experiments. Thus, the importance of the CST data in the estimation of parameters of the thermodynamic model is confirmed through this work.

Keywords: concentrated electrolytes, Debye-Hückel theory, interaction parameters, Robinson-Stokes-Glueckauf model, Flory-Huggins model, critical solution temperature

Procedia PDF Downloads 355

Authors: Yi H. Wong, Li L. Chan, Chee O. Leong, Stephen Ambu, Joon W. Mak, Priyadashi S. Sahu

Abstract:

Background: Acanthamoeba is a free-living opportunistic protist which is ubiquitously distributed in the environment. Virulent Acanthamoeba can cause fatal encephalitis in immunocompromised patients and potential blinding keratitis in immunocompetent contact lens wearers. Approximately 24 species have been identified but only the A. castellanii, A. polyphaga and A. culbertsoni are commonly associated with human infections. Until to date, the precise molecular basis for Acanthamoeba pathogenesis remains unclear. Previous studies reported that Acanthamoeba virulence can be diminished through prolonged axenic culture but revived through serial mouse passages. As no clear explanation on this reversible pathogenesis is established, hereby, we postulate that the epigenetic regulators, DNA-methyltransferases (DNMT) and histone-deacetylases (HDAC), could possibly be involved in granting the virulence plasticity of Acanthamoeba spp. Methods: Four rounds of mouse passages were conducted to revive the virulence potential of the virulence-attenuated Acanthamoeba castellanii strain (ATCC 50492). Briefly, each mouse (n=6/group) was inoculated intraperitoneally with Acanthamoebae cells (2x 105 trophozoites/mouse) and incubated for 2 months. Acanthamoebae cells were isolated from infected mouse organs by culture method and subjected to subsequent mouse passage. In vitro cytopathic, encystment and gelatinolytic assays were conducted to evaluate the virulence characteristics of Acanthamoebae isolates for each passage. PCR primers which targeted on the 2 members (DNMT1 and DNMT2) and 5 members (HDAC1 to 5) of the DNMT and HDAC gene families respectively were custom designed. Quantitative real-time PCR (qPCR) was performed to detect and quantify the relative expression of the two gene families in each Acanthamoeba isolates. Beta-tubulin of A. castellanii (Genbank accession no: XP_004353728) was included as housekeeping gene for data normalisation. PCR mixtures were also analyzed by electrophoresis for amplicons detection. All statistical analyses were performed using the paired one-tailed Student’s t test. Results: Our pathogenicity tests showed that the virulence-reactivated Acanthamoeba had a higher degree of cytopathic effect on vero cells, a better resistance to encystment challenge and a higher gelatinolytic activity which was catalysed by serine protease. qPCR assay showed that DNMT1 expression was significantly higher in the virulence-reactivated compared to the virulence-attenuated Acanthamoeba strain (p ≤ 0.01). The specificity of primers which targeted on DNMT1 was confirmed by sequence analysis of PCR amplicons, which showed a 97% similarity to the published DNA-methyltransferase gene of A. castellanii (GenBank accession no: XM_004332804.1). Out of the five primer pairs which targeted on the HDAC family genes, only HDAC4 expression was significantly difference between the two variant strains. In contrast to DNMT1, HDAC4 expression was much higher in the virulence-attenuated Acanthamoeba strain. Conclusion: Our mouse passages had successfully restored the virulence of the attenuated strain. Our findings suggested that DNA-methyltransferase (DNMT1) and histone deacetylase (HDAC4) expressions are associated with virulence potential of Acanthamoeba spp.

Keywords: acanthamoeba, DNA-methyltransferase, histone deacetylase, virulence-associated proteins

Procedia PDF Downloads 259

Authors: Yi-Hao Wong, Li-Li Chan, Chee-Onn Leong, Stephen Ambu, Joon-Wah Mak, Priyasashi Sahu

Abstract:

Background: Acanthamoeba is a genus of amoebae which lives as a free-living in nature or as a human pathogen that causes severe brain and eye infections. Virulence potential of Acanthamoeba is not constant and can change with growth conditions. DNA methylation, an epigenetic process which adds methyl groups to DNA, is used by eukaryotic cells, including several human parasites to control their gene expression. We used qPCR, siRNA gene silencing, and RNA sequencing (RNA-Seq) to study DNA-methyltransferase gene family (DNMT) in order to indicate the possibility of its involvement in programming Acanthamoeba virulence potential. Methods: A virulence-attenuated Acanthamoeba isolate (designation: ATCC; original isolate: ATCC 50492) was subjected to mouse passages to restore its pathogenicity; a virulence-reactivated isolate (designation: AC/5) was generated. Several established factors associated with Acanthamoeba virulence phenotype were examined to confirm the succession of reactivation process. Differential gene expression of DNMT between ATCC and AC/5 isolates was performed by qPCR. Silencing on DNMT gene expression in AC/5 isolate was achieved by siRNA duplex. Total RNAs extracted from ATCC, AC/5, and siRNA-treated (designation: si-146) were subjected to RNA-Seq for comparative transcriptomic analysis in order to identify the genome-wide effect of DNMT in regulating Acanthamoeba gene expression. qPCR was performed to validate the RNA-Seq results. Results: Physiological and cytophatic assays demonstrated an increased in virulence potential of AC/5 isolate after mouse passages. DNMT gene expression was significantly higher in AC/5 compared to ATCC isolate (p ≤ 0.01) by qPCR. si-146 duplex reduced DNMT gene expression in AC/5 isolate by 30%. Comparative transcriptome analysis identified the differentially expressed genes, with 3768 genes in AC/5 vs ATCC isolate; 2102 genes in si-146 vs AC/5 isolate and 3422 genes in si-146 vs ATCC isolate, respectively (fold-change of ≥ 2 or ≤ 0.5, p-value adjusted (padj) < 0.05). Of these, 840 and 1262 genes were upregulated and downregulated, respectively, in si-146 vs AC/5 isolate. Eukaryotic orthologous group (KOG) assignments revealed a higher percentage of downregulated gene expression in si-146 compared to AC/5 isolate, were related to posttranslational modification, signal transduction and energy production. Gene Ontology (GO) terms for those downregulated genes shown were associated with transport activity, oxidation-reduction process, and metabolic process. Among these downregulated genes were putative genes encoded for heat shock proteins, transporters, ubiquitin-related proteins, proteins for vesicular trafficking (small GTPases), and oxidoreductases. Functional analysis of similar predicted proteins had been described in other parasitic protozoa for their survival and pathogenicity. Decreased expression of these genes in si146-treated isolate may account in part for Acanthamoeba reduced pathogenicity. qPCR on 6 selected genes upregulated in AC/5 compared to ATCC isolate corroborated the RNA sequencing findings, indicating a good concordance between these two analyses. Conclusion: To the best of our knowledge, this study represents the first genome-wide analysis of DNA methylation and its effects on gene expression in Acanthamoeba spp. The present data indicate that DNA methylation has substantial effect on global gene expression, allowing further dissection of the genome-wide effects of DNA-methyltransferase gene in regulating Acanthamoeba pathogenicity.

Keywords: Acanthamoeba, DNA methylation, RNA sequencing, virulence

Procedia PDF Downloads 169