Search results for: Neeraj Sahu

Authors: Satish Kumar, Spandan Sahu, Sarjati Sahoo, K. K. Khatua

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An experimental investigation has been carried out in a tilting flume of 2 m wide, 13 m long, and 0.3 m deep to study the effect of flow on the formation of braided channel pattern. Sediment flow is recirculated through the flume, which passes from the headgate to the sediment/water collecting tank through the tailgate. Further, without altering the geometry of the sand bed channel, the discharge is varied to study the effect of the formation of the braided pattern with time. Then the flow rate is varied to study the effect of flow on the formation of the braided pattern. Sediment transport rate is highly variable and was found to be a nonlinear function of flow rate, aspect ratio, longitudinal slope, and time. Total braided intensity (BIT) for each discharge case is found to be more than the active braided intensity (BIA). Both the parameters first increase and then decrease as the time progresses following a similar pattern for all the observed discharge cases. When the flow is increased, the movement of sediment also increases since the active braided intensity is found to adjust quickly. The measurement of velocity and boundary shear helps to study the erosion and sedimentation processes in the channel and formation of small meandering channels and then the braided channel for different discharge conditions of a sediment river. Due to regime properties of rivers, both total braided Intensity and active braided intensity become stable for a given channel and flow conditions. In the present case, the trend of the ratio of BIA to BIT is found to be asymptotic against the time with a value of 0.4. After the particular time elapses off the flow, new small channels are also found to be formed with changes in the sinuosity of the active channels, thus forming the braided network. This is due to the continuous erosion and sedimentation processes occurring for the flow process for the flow and sediment conditions.

Keywords: active braided intensity, bed load, sediment transport, shear stress, total braided intensity

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Authors: Atma Sahu

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After 9/11, there will only be cyberwars. The cyberwars increase in intensity the country's cybersecurity workforce's hiring and retention issues. Currently, many organizations have unfilled cybersecurity positions, and to a lesser degree, their cybersecurity teams are understaffed. Therefore, there is a critical need to develop a new program to help meet the market demand for cybersecurity engineers (CYSE) and personnel. Coppin State University in the United States was responsible for developing a cybersecurity engineering BS degree program. The CYSE curriculum design methodology consisted of three parts. First, the ACM Cross-Cutting Concepts standard's pervasive framework helped curriculum designers and students explore connections among the core courses' knowledge areas and reinforce the security mindset conveyed in them. Second, the core course context was created to assist students in resolving security issues in authentic cyber situations involving cyber security systems in various aspects of industrial work while adhering to the NIST standards framework. The last part of the CYSE curriculum design aspect was the institutional student learning outcomes (SLOs) integrated and aligned in content courses, representing more detailed outcomes and emphasizing what learners can do over merely what they know. The CYSE program's core courses express competencies and learning outcomes using action verbs from Bloom's Revised Taxonomy. This aspect of the CYSE BS degree program's design is based on these three pillars: the ACM, NIST, and SLO standards, which all CYSE curriculum designers should know. This unique CYSE curriculum design methodology will address how students and the CYSE program will be assessed and evaluated. It is also critical that educators, program managers, and students understand the importance of staying current in this fast-paced CYSE field.

Keywords: cyber security, cybersecurity engineering, systems engineering, NIST standards, physical systems

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Authors: Satpal Yadav, Ashish Phulkar, Krishna K. Sahu

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Aim: The research aims at doing a 3 Dimension biomechanical analysis in the shot put techniques of International throwers to evaluate the performance. Research Method: The researcher adopted the descriptive method and the data was subjected to calculate by using Pearson’s product moment correlation for the correlation of the biomechanical parameters with the performance of shot put throw. In all the analyses, the 5% critical level (p ≤ 0.05) was considered to indicate statistical significance. Research Sample: Eight (N=08) international shot putters using rotational/glide technique in male category was selected as subjects for the study. The researcher used the following methods and tools to obtain reliable measurements the instrument which was used for the purpose of present study namely the tesscorn slow-motion camera, specialized motion analyzer software, 7.260 kg Shot Put (for a male shot-putter) and steel tape. All measurement pertaining to the biomechanical variables was taken by the principal investigator so that data collected for the present study was considered reliable. Results: The finding of the study showed that negative significant relationship between the angular velocity right shoulder, acceleration distance at pre flight (-0.70), (-0.72) respectively were obtained, the angular displacement of knee, angular velocity right shoulder and acceleration distance at flight (0.81), (0.75) and (0.71) respectively were obtained, the angular velocity right shoulder and acceleration distance at transition phase (0.77), (0.79) respectively were obtained and angular displacement of knee, angular velocity right shoulder, release velocity shot, angle of release, height of release, projected distance and measured distance as the values (0.76), (0.77), (-0.83), (-0.79), (-0.77), (0.99) and (1.00) were found higher than the tabulated value at 0.05 level of significance. On the other hand, there exists an insignificant relationship between the performance of shot put and acceleration distance [m], angular displacement shot, C.G at release and horizontal release distance on the technique of shot put.

Keywords: biomechanics, analysis, shot put, international throwers

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Authors: Pabitra Sahu, M. Manohari, S. Priyadharshini, R. Santhanam, S. Chandrasekaran, B. Venkatraman

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Radiation workers of reprocessing plants have a potential for internal exposure due to actinides and fission products. Radionuclides like Americium, lead, Polonium and Europium are bone seekers and get accumulated in the skeletal part. As the major skeletal content is in the skull (13%) and knee (22%), measurements of old intake have to be carried out in the skull and knee. At the Indira Gandhi Centre for Atomic Research, a twin HPGe-based actinide monitor is used for the measurement of actinides present in bone. Efficiency estimation, which is one of the prerequisites for the quantification of radionuclides, requires anthropomorphic phantoms. Such phantoms are very limited. Hence, in this study, efficiency curves for a Twin HPGe-based actinide monitoring system are established theoretically using the FLUKA Monte Carlo method and ICRP adult male voxel phantom. In the case of skull measurement, the detector is placed over the forehead, and for knee measurement, one detector is placed over each knee. The efficiency values of radionuclides present in the knee and skull vary from 3.72E-04 to 4.19E-04 CPS/photon and 5.22E-04 to 7.07E-04 CPS/photon, respectively, for the energy range 17 to 3000keV. The efficiency curves for the measurement are established, and it is found that initially, the efficiency value increases up to 100 keV and then starts decreasing. It is found that the skull efficiency values are 4% to 63% higher than that of the knee, depending on the energy for all the energies except 17.74 keV. The reason is the closeness of the detector to the skull compared to the knee. But for 17.74 keV the efficiency of the knee is more than the skull due to the higher attenuation caused in the skull bones because of its greater thickness. The Minimum Detectable Activity (MDA) for 241Am present in the skull and knee is 9 Bq. 239Pu has a MDA of 950 Bq and 1270 Bq for knee and skull, respectively, for a counting time of 1800 sec. This paper discusses the simulation method and the results obtained in the study.

Keywords: FLUKA Monte Carlo Method, ICRP adult male voxel phantom, knee, Skull.

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Authors: Vaishali Patil, Neeraj Masand

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In older people, Alzheimer’s disease (AD) is turning out to be a lethal disease. According to the amyloid hypothesis, aggregation of the amyloid β–protein (Aβ), particularly its 42-residue variant (Aβ42), plays direct role in the pathogenesis of AD. Aβ is generated through sequential cleavage of amyloid precursor protein (APP) by β–secretase (BACE) and γ–secretase (GS). Thus in the treatment of AD, γ-secretase modulators (GSMs) are potential disease-modifying as they selectively lower pathogenic Aβ42 levels by shifting the enzyme cleavage sites without inhibiting γ–secretase activity. This possibly avoids known adverse effects observed with complete inhibition of the enzyme complex. Virtual screening, via drug-like ADMET filter, QSAR and molecular docking analyses, has been utilized to identify novel γ–secretase modulators with sulphonamide nucleus. Based on QSAR analyses and docking score, some novel analogs have been synthesized. The results obtained by in silico studies have been validated by performing in vivo analysis. In the first step, behavioral assessment has been carried out using Scopolamine induced amnesia methodology. Later the same series has been evaluated for neuroprotective potential against the oxidative stress induced by Scopolamine. Biochemical estimation was performed to evaluate the changes in biochemical markers of Alzheimer’s disease such as lipid peroxidation (LPO), Glutathione reductase (GSH), and Catalase. The Scopolamine induced amnesia model has shown increased Acetylcholinesterase (AChE) levels and the inhibitory effect of test compounds in the brain AChE levels have been evaluated. In all the studies Donapezil (Dose: 50µg/kg) has been used as reference drug. The reduced AChE activity is shown by compounds 3f, 3c, and 3e. In the later stage, the most potent compounds have been evaluated for Aβ42 inhibitory profile. It can be hypothesized that this series of alkyl-aryl sulphonamides exhibit anti-AD activity by inhibition of Acetylcholinesterase (AChE) enzyme as well as inhibition of plaque formation on prolong dosage along with neuroprotection from oxidative stress.

Keywords: gamma-secretase inhibitors, Alzzheimer's disease, sulphonamides, QSAR

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Authors: Malay K. Das, Bhanu P. Sahu

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Furosemide is a weakly acidic diuretic indicated for treatment of edema and hypertension. It has very poor solubility but high permeability through stomach and upper gastrointestinal tract (GIT). Due to its limited solubility it has poor and variable oral bioavailability of 10-90%. The aim of this study was to enhance the oral bioavailability of furosemide by preparation of nanosuspensions. The nanosuspensions were prepared by nanoprecipitation with sonication using DMSO (dimethyl sulfoxide) as a solvent and water as an antisolvent (NA). The prepared nanosuspensions were sterically stabilized with polyvinyl acetate (PVA).These were characterized for particle size, ζ potential, polydispersity index, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD) pattern and release behavior. The effect of nanoprecipitation on oral bioavailability of furosemide nanosuspension was studied by in vitro dissolution and in vivo absorption study in rats and compared to pure drug. The stable nanosuspension was obtained with average size range of the precipitated nanoparticles between 150-300 nm and was found to be homogenous showing a narrow polydispersity index of 0.3±0.1. DSC and XRD studies indicated that the crystalline furosemide drug was converted to amorphous form upon precipitation into nanoparticles. The release profiles of nanosuspension formulation showed up to 81.2% release in 4 h. The in vivo studies on rats revealed a significant increase in the oral absorption of furosemide in the nanosuspension compared to pure drug. The AUC0→24 and Cmax values of nanosuspension were approximately 1.38 and 1.68-fold greater than that of pure drug, respectively. Furosemide nanosuspension showed 20.06±0.02 % decrease in systolic blood pressure compared to 13.37±0.02 % in plain furosemide suspension, respectively. The improved oral bioavailability and pharmacodynamics effect of furosemide may be due to the improved dissolution of furosemide in simulated gastric fluid which results in enhanced oral systemic absorption of furosemide from stomach region where it has better permeability.

Keywords: furosemide, nanosuspension, bioavailability enhancement, nanoprecipitation, oral drug delivery

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Authors: Abhilash Sahu, Amit Priyadarshi

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In this paper, we will discuss the existence of weak solutions of the Kirchhoff type boundary value problem on the Sierpinski gasket. Where S denotes the Sierpinski gasket in R² and S₀ is the intrinsic boundary of the Sierpinski gasket. M: R → R is a positive function and h: S × R → R is a suitable function which is a part of our main equation. ∆p denotes the p-Laplacian, where p > 1. First of all, we will define a weak solution for our problem and then we will show the existence of at least two solutions for the above problem under suitable conditions. There is no well-known concept of a generalized derivative of a function on a fractal domain. Recently, the notion of differential operators such as the Laplacian and the p-Laplacian on fractal domains has been defined. We recall the result first then we will address the above problem. In view of literature, Laplacian and p-Laplacian equations are studied extensively on regular domains (open connected domains) in contrast to fractal domains. In fractal domains, people have studied Laplacian equations more than p-Laplacian probably because in that case, the corresponding function space is reflexive and many minimax theorems which work for regular domains is applicable there which is not the case for the p-Laplacian. This motivates us to study equations involving p-Laplacian on the Sierpinski gasket. Problems on fractal domains lead to nonlinear models such as reaction-diffusion equations on fractals, problems on elastic fractal media and fluid flow through fractal regions etc. We have studied the above p-Laplacian equations on the Sierpinski gasket using fibering map technique on the Nehari manifold. Many authors have studied the Laplacian and p-Laplacian equations on regular domains using this Nehari manifold technique. In general Euler functional associated with such a problem is Frechet or Gateaux differentiable. So, a critical point becomes a solution to the problem. Also, the function space they consider is reflexive and hence we can extract a weakly convergent subsequence from a bounded sequence. But in our case neither the Euler functional is differentiable nor the function space is known to be reflexive. Overcoming these issues we are still able to prove the existence of at least two solutions of the given equation.

Keywords: Euler functional, p-Laplacian, p-energy, Sierpinski gasket, weak solution

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Authors: S. K. Dewangan, K. R. Sahu, S. Soni

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Unemployment is today’s basic socio-economic problem eroding national income and living standards, aggravating national development and poverty alleviation. The farmers are encouraged to take up non-agriculture practices which are integrated with Sericulture. Sericulture is one of the primary occupations for livelihood of poor people in tribal area. Most of tribal are involved in Sericulture. Tasar, Eri are the main forest-based cultivation. Among these sericultures is the major crop adopted by the Tribal’s and practiced in respective areas. Out of the 6, 38,588 villages in India, sericultures are practiced in about 69000 villages providing employment to about 7.85 million people. Sericulture is providing livelihood for 9, 47,631 families. India continues to be the second largest producer of silk in the World. Among the four varieties of silk produced, as in 2012-13, Mulberry accounts for 18,715 MT, Eri 3116 MT, Tasar 1729 MT and Muga 119MT of the total raw silk production in the country. Sericulture with its unique features plays an important role in upgrading the socio-economic conditions of the rural folk and with employment opportunities to the educated rural youth and women. In view of the importance of sericulture enterprise for the biodiversity conservation as well as its cultural bondage, the paper tries to enlighten and discuss the significance of sericulture and strategies to be taken for the employment generation in Indian sericulture industry. The present paper explores the possible employment opportunities derived from problem analysis and strategies to be adopted aiming at revolutionary biodiversity conservation in the study area. The paper highlights the sericulture is a way for biodiversity conservation, employment generation in Raigarh district, their utilization and needs as they act as a tool for socio-economic change for tribal. The study concludes with some suggestions to improve the feasibility of sericulture in long term.

Keywords: bio-diversity, employment, sericulture, tribal, income, socio-economic

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Authors: Spandan Sahu, Amiya Kumar Pati, Kishanjit Kumar Khatua

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Vegetation plays a major role in deciding the flow parameters in an open channel. It enhances the aesthetic view of the revetments. The major types of vegetation in river typically comprises of herbs, grasses, weeds, trees, etc. The vegetation in an open channel usually consists of aquatic plants with complete submergence, partial submergence, floating plants. The presence of vegetative plants can have both benefits and problems. The major benefits of aquatic plants are they reduce the soil erosion, which provides the water with a free surface to move on without hindrance. The obvious problems are they retard the flow of water and reduce the hydraulic capacity of the channel. The degree to which the flow parameters are affected depends upon the density of the vegetation, degree of submergence, pattern of vegetation, vegetation species. Vegetation in open channel tends to provide resistance to flow, which in turn provides a background to study the varying trends in flow parameters having vegetative growth in the channel surface. In this paper, an experiment has been conducted on a meandering channel having sinuosity of 1.33 with rigid vegetation cover to investigate the effect on flow parameters, variation of manning’s n with degree of the denseness of vegetation, vegetation pattern and submergence criteria. The measurements have been carried out in four different cross-sections two on trough portion of the meanders, two on the crest portion. In this study, the analytical solution of Shiono and knight (SKM) for lateral distributions of depth-averaged velocity and bed shear stress have been taken into account. Dimensionless eddy viscosity and bed friction have been incorporated to modify the SKM to provide more accurate results. A mathematical model has been formulated to have a comparative analysis with the results obtained from Shiono-Knight Method.

Keywords: bed friction, depth averaged velocity, eddy viscosity, SKM

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Authors: Arun Kharate, Sarata Kumar Sahu, Susen Kumar Panda, Niranjan Sahoo, H. K. Panda

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In the present study, an attempt has been made for isolation and identification of feline panleukopaenia virus (FPLV) from wild felids of Nandankanan zoo, Odisha, India, along with prevalence study of FPLV. Fecal samples collected from wild felids (26 tigers, 22 lions, 5 leopards, 3 hyenas, 1 jaguar, 2 foxes and 1 wild cat) were subjected to hemagglutinnation test and fluorescent antibody test. In hemagglutinnation test 13 (50%) samples from tiger, 14 (63.63%) samples from lions, 1 (20%) sample from leopards, 1 (50%) from fox, 3 (100%) samples from hyenas and 1 (100%) sample from wild cat were positive. On fluorescent antibody test (FAT), 15 (57.69%) samples from tiger, 18 (81.81%) from lions, 2 (40%) from leopards, 1 (50%) from fox, 3 (100%) from hyenas and 1 (100%) from wild cat were positive. FPLV was isolated using MDBK cell line and preliminary characterization was done on the basis of characteristic cytopathic effect. The virus samples were quantified through titration in MDBK cells. Serological confirmation of FPLV isolates was carried out by HI test, micro-SNT and indirect-ELISA. Physico-chemical characters like pH and temperature resistance along molecular identification using specific FPLV primers was carried out. Seroprevalence study of 36 serum samples employing HI test, micro SNT and indirect-ELISA revealed prevalence of 38.8, 44.4 and 72.2% respectively. During study period an adult tigress and a tiger cub died suspected of feline panleukopenia. The necropsy findings in both animals showed hemorrhagic gastroenteritis. The cytological examination revealed presence of intranuclear inclusion bodies in the intestinal epithelial cells. Spleen, mesenteric lymph node and intestine were positive for feline panleukopenia by FAT. The investigation revealed that feline panleukopenia was prevalent in wild felines of Nandankanan zoo.

Keywords: Feline panleukopenia, fluorescent antibody test, hemagglutination test, indirect-ELISA, Nandankanan zoo

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Authors: Ranjit Kumar Sahu, Ramakar Jha

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Urban Water Management is the practice of managing freshwater, waste water, and storm water as components of a basin-wide management plan. It builds on existing water supply and sanitation considerations within an urban settlement by incorporating urban water management within the scope of the entire river basin. The pervasive problems generated by urban development have prompted, in the present work, to study the spatial extent of urbanization in Golden Triangle of Odisha connecting the cities Bhubaneswar (20.2700° N, 85.8400° E), Puri (19.8106° N, 85.8314° E) and Konark (19.9000° N, 86.1200° E)., and patterns of periodic changes in urban development (systematic/random) in order to develop future plans for (i) urbanization promotion areas, and (ii) urbanization control areas. Remote Sensing, using USGS (U.S. Geological Survey) Landsat8 maps, supervised classification of the Urban Sprawl has been done for during 1980 - 2014, specifically after 2000. This Work presents the following: (i) Time series analysis of Hydrological data (ground water and rainfall), (ii) Application of SWMM (Storm Water Management Model) and other soft computing techniques for Urban Water Management, and (iii) Uncertainty analysis of model parameters (Urban Sprawl and correlation analysis). The outcome of the study shows drastic growth results in urbanization and depletion of ground water levels in the area that has been discussed briefly. Other relative outcomes like declining trend of rainfall and rise of sand mining in local vicinity has been also discussed. Research on this kind of work will (i) improve water supply and consumption efficiency (ii) Upgrade drinking water quality and waste water treatment (iii) Increase economic efficiency of services to sustain operations and investments for water, waste water, and storm water management, and (iv) engage communities to reflect their needs and knowledge for water management.

Keywords: Storm Water Management Model (SWMM), uncertainty analysis, urban sprawl, land use change

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Authors: Baikunthanath Sahoo, Santosh Kumar Sahu, Krishna Malakar

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With the essence of global environmental sustainability and green business management, the wind of business research moved towards Corporate Social Responsibility. In addition to international and national treaties, businesses have also started realising environmental protection and energy efficiency through CSR as part of business strategy in response to climate change. Considering the ambitious emission reduction target and rapid economic development of India, this study is an attempt to explore the effect of CSR on the energy efficiency management of manufacturing firms in India. By using firm-level data, the panel fixed effect model shows that the CSR dummy variable is negatively influencing the energy intensity or technically, they are energy efficient. The result demonstrates that in the presence of CSR, all the production economic variables are significant. The result also shows that doing environmental expenditure does not improve energy efficiency might be because very few firms are motivated to do such expenditure and also not common to all sectors. The interactive effect model result conforms that without considering CSR dummy as an intervening variable only Manufacturers of Chemical and Chemical products, Manufacturers of Pharmaceutical, medical chemical, and botanical products firms energy intensity low but after considering CSR in their business practices all six sub-sector firms become energy efficient. The empirical result also validate that firms are continuously engaged in CSR activities they are highly energy efficient. It is an important motivational factor for firms to become economically and environmentally sustainable in the corporate world. This analysis would help business practitioners to know how to manage today’s profitability and tomorrow’s sustainability to achieve a comparative advantage in the emerging market economy. The paper concludes that reducing energy consumption as part of their social responsibility to care for the environment, will need collaborative efforts of business society and policy bodies.

Keywords: CSR, Energy Efficiency, Indian manufacturing Sector, Business strategy

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Authors: Pushkar Lal Sharma, Sanjana Singh, Umesh Kumar Sahu

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This paper helps to understand the need of Customer Relationship Management in Libraries and why Librarians should implement the marketing concept of Customer Relationship Management in their libraries. As like any industry, libraries too face growing challenges to continuously meet customer expectations, and attract and retain users in light of overflowing competition. The ability to understand customers, build relationships and market diverse services is essential when considering ways to expand service offerings and improve Return on Investment. Since Library is service oriented Enterprise, hence the Customer/User/ Reader/Patron are the most important element of Library & Information System to whom and for whom library offers various services. How to provide better and most efficient services to its users is the main concern of every Library & Information centre in the present era. The basic difference between Business Enterprise and Library Information System is that ‘in Business System ‘the efficiency is measured in terms of ’profit’ or ‘monetary gains’; whereas in a Library & Information System, the efficiency is measured in terms of ‘services’ and therefore the goals that are set in Business Enterprise are’ profit oriented’ whereas goals set in the Library & Information Centre are ‘Service-oriented’. With the explosion of information and advancement of technology readers have so many choices to get information rather than visiting a library. Everything is available at the click of a mouse, library customers have become more knowledgeable and demanding in an era marked by abundance of information resources and services. With this explosion of information in every field of knowledge and choice in selection of service, satisfying user has become a challenge now a day for libraries. Accordingly, Libraries have to build good relationship with its users by adopting Customer relationship Management. CRM refers to the methods and tools which help an organization to manage its relationship with its customers in an organized way. The Customer Relationship Management (CRM) combines business strategy and technology to identify, acquire and retain good customer relationship. The goal of CRM is to optimize management of customer information needs & interests and increase customer satisfaction and loyalty. Implementing CRM in Libraries can improve customer data and process management, customer loyalty, retention and satisfaction.

Keywords: customer relationship management, CRM, CRM tools, customer satisfaction

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Authors: Anamika Sahu

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The Himalayas, like many mountainous regions, is susceptible to multiple hazards. In recent times, the frequency of such disasters is continuously increasing due to extreme weather phenomena. These natural hazards are responsible for irreparable human and economic loss. The Indian Himalayas has repeatedly been ruptured by great earthquakes in the past and has the potential for a future large seismic event as it falls under the seismic gap. Damages caused by earthquakes are different in different localities. It is well known that, during earthquakes, damage to the structure is associated with the subsurface conditions and the quality of construction materials. So, for sustainable mountain development, prior estimation of site characterization will be valuable for designing and constructing the space area and for efficient mitigation of the seismic risk. Both geotechnical and geophysical investigation of the subsurface is required to describe the subsurface complexity. In mountainous regions, geophysical methods are gaining popularity as areas can be studied without disturbing the ground surface, and also these methods are time and cost-effective. The MASW method is used to calculate the Vs30. Vs30 is the average shear wave velocity for the top 30m of soil. Shear wave velocity is considered the best stiffness indicator, and the average of shear wave velocity up to 30 m is used in National Earthquake Hazards Reduction Program (NEHRP) provisions (BSSC,1994) and Uniform Building Code (UBC), 1997 classification. Parameters obtained through geotechnical investigation have been integrated with findings obtained through the subsurface geophysical survey. Joint interpretation has been used to establish inter-relationships among mineral constituents, various textural parameters, and unconfined compressive strength (UCS) with shear wave velocity. It is found that results obtained through the MASW method fitted well with the laboratory test. In both conditions, mineral constituents and textural parameters (grain size, grain shape, grain orientation, and degree of interlocking) control the petrophysical and mechanical properties of rocks and the behavior of shear wave velocity.

Keywords: MASW, mechanical, petrophysical, site characterization

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Authors: Arpit Kumar Shrivastava, Subrat Kumar, Rajani Kanta Mohapatra, Priyadarshi Soumyaranjan Sahu

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Computational approaches to predict structure, function and other biological characteristics of proteins are becoming more common in comparison to the traditional methods in drug discovery. Cryptosporidiosis is a major zoonotic diarrheal disease particularly in children, which is caused primarily by Cryptosporidium hominis and Cryptosporidium parvum. Currently, there are no vaccines for cryptosporidiosis and recommended drugs are not effective. With the availability of complete genome sequence of C. hominis, new targets have been recognized for the development of effective and better drugs and/or vaccines. We identified a unique hypothetical epitopic protein in C. hominis genome through BLASTP analysis. A 3D model of the hypothetical protein was generated using I-Tasser server through threading methodology. The quality of the model was validated through Ramachandran plot by PROCHECK server. The functional annotation of the hypothetical protein through DALI server revealed structural similarity with human Transportin 3. Phylogenetic analysis for this hypothetical protein also showed C. hominis hypothetical protein (CUV04613) was the closely related to human transportin 3 protein. The 3D protein model is further subjected to virtual screening study with inhibitors from the Zinc Database by using Dock Blaster software. Docking study reported N-(3-chlorobenzyl) ethane-1,2-diamine as the best inhibitor in terms of docking score. Docking analysis elucidated that Leu 525, Ile 526, Glu 528, Glu 529 are critical residues for ligand–receptor interactions. The molecular dynamic simulation was done to access the reliability of the binding pose of inhibitor and protein complex using GROMACS software at 10ns time point. Trajectories were analyzed at each 2.5 ns time interval, among which, H-bond with LEU-525 and GLY- 530 are significantly present in MD trajectories. Furthermore, antigenic determinants of the protein were determined with the help of DNA Star software. Our study findings showed a great potential in order to provide insights in the development of new drug(s) or vaccine(s) for control as well as prevention of cryptosporidiosis among humans and animals.

Keywords: cryptosporidium hominis, hypothetical protein, molecular docking, molecular dynamics simulation

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Authors: Amiya Kumar Pati, Spandan Sahu, Kishanjit Kumar Khatua

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River is our main source of water which is a form of open channel flow and the flow in the open channel provides with many complex phenomena of sciences that needs to be tackled such as the critical flow conditions, boundary shear stress, and depth-averaged velocity. The development of society, more or less solely depends upon the flow of rivers. The rivers are major sources of many sediments and specific ingredients which are much essential for human beings. A river flow consisting of small and shallow channels sometimes divide and recombine numerous times because of the slow water flow or the built up sediments. The pattern formed during this process resembles the strands of a braid. Braided streams form where the sediment load is so heavy that some of the sediments are deposited as shifting islands. Braided rivers often exist near the mountainous regions and typically carry coarse-grained and heterogeneous sediments down a fairly steep gradient. In this paper, the apparent shear stress formulae were suitably modified, and the Energy Concept Method (ECM) was applied for the prediction of discharges at the junction of a two-flow braided compound channel. The Energy Concept Method has not been applied for estimating the discharges in the braided channels. The energy loss in the channels is analyzed based on mechanical analysis. The cross-section of channel is divided into two sub-areas, namely the main-channel below the bank-full level and region above the bank-full level for estimating the total discharge. The experimental data are compared with a wide range of theoretical data available in the published literature to verify this model. The accuracy of this approach is also compared with Divided Channel Method (DCM). From error analysis of this method, it is observed that the relative error is less for the data-sets having smooth floodplains when compared to rough floodplains. Comparisons with other models indicate that the present method has reasonable accuracy for engineering purposes.

Keywords: critical flow, energy concept, open channel flow, sediment, two-flow braided compound channel

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Authors: S. Vaibhav Kant Sahu, Surabhi Bipin Seth

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Human well-being has multiple constituents including the basic material for a good life, freedom and choice, health, good social relations, and security. Poverty is also multidimensional and has been defined as the pronounced deprivation of well-being. Dhurwa tribe of Bastar (India) have symbiotic relation with nature, it provisions ecosystem service such as food, fuel and fiber; regulating services such as climate regulation and non-material benefits such as spiritual or aesthetic benefits and they are managing their forest from ages. The demand for ecosystem services is now so great that trade-off among services become rule. Aim of study to explore evidences for linkages between ecosystem services and well-being of indigenous community, how much it helps them in poverty reduction and interaction between them. Objective of study was to find drivers of change and evidence concerning link between ecosystem, human development and sustainability, evidence in decision making does it opt for multi sectoral objectives. Which means human well-being as the central focus for assessment, while recognizing that biodiversity and ecosystems also have intrinsic value. Ecosystem changes that may have little impact on human well-being over days or weeks may have pronounced impacts over years or decades; so assessments needed to be conducted at spatial and temporal scales under social, political, economic scales to have high-resolution data. Researcher used framework developed by Millennium ecosystem assessment; since human action now directly or unknowingly virtually alter ecosystem. Researcher used ethnography study to get primary qualitative data, secondary data collected from panchayat office. The responses were transcribed and translated into English, as interview held in Hindi and local indigenous language. Focus group discussion were held with group of 10 women at Tiriya village. Researcher concluded with well-being is not just gap between ecosystem service supply but also increases vulnerability. Decision can have consequences external to the decision framework these consequences are called externalities because they are not part of the decision-making calculus.

Keywords: Bastar, Dhurwa tribe, ecosystem services, millennium ecosystem assessment, sustainability

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Authors: Neeraj Vij, Christian Leber, Kenneth Schmidt

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Introduction: Total knee arthroplasty is a common procedure. It is well known that the biomechanics of the knee do not fully return to their normal state. Motion analysis has been used to study the biomechanics of the knee after total knee arthroplasty. The purpose of this scoping review is to summarize the current use of gait analysis in total knee arthroplasty and to identify the preoperative motion analysis parameters for which a systematic review aimed at determining the reliability and validity may be warranted. Materials and Methods: This IRB-exempt scoping review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) checklist strictly. Five search engines were searched for a total of 279 articles. Articles underwent a title and abstract screening process followed by full-text screening. Included articles were placed in the following sections: the role of gait analysis as a research tool for operative decisions, other research applications for motion analysis in total knee arthroplasty, gait analysis as a tool in predicting radiologic outcomes, gait analysis as a tool in predicting clinical outcomes. Results: Eleven articles studied gait analysis as a research tool in studying operative decisions. Motion analysis is currently used to study surgical approaches, surgical techniques, and implant choice. Five articles studied other research applications for motion analysis in total knee arthroplasty. Other research applications for motion analysis currently include studying the role of the unicompartmental knee arthroplasty and novel physical therapy protocols aimed at optimizing post-operative care. Two articles studied motion analysis as a tool for predicting radiographic outcomes. Preoperative gait analysis has identified parameters than can predict postoperative tibial component migration. 15 articles studied motion analysis in conjunction with clinical scores. Conclusions: There is a broad range of applications within the research domain of total knee arthroplasty. The potential application is likely larger. However, the current literature is limited by vague definitions of ‘gait analysis’ or ‘motion analysis’ and a limited number of articles with preoperative and postoperative functional and clinical measures. Knee adduction moment, knee adduction impulse, total knee range of motion, varus angle, cadence, stride length, and velocity have the potential for integration into composite clinical scores. A systematic review aimed at determining the validity, reliability, sensitivities, and specificities of these variables is warranted.

Keywords: motion analysis, joint replacement, patient-reported outcomes, knee surgery

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Authors: Atul C. Mahajan, A. K. Chakravarty, V. Jamuna, C. S. Patil, Neeraj Kashyap, Bharti Deshmukh, Vijay Kumar

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The selection of Sahiwal bulls on the basis of dams best lactation milk yield under breeding programme in herd of the country neglecting fertility traits leads to deterioration in their performances and economy. The goal of this study was to explore polymorphism of CRISP2 gene and their association with semen traits (Post Thaw Motility, Hypo-osmotic Swelling Test, Acrosome Integrity, DNA Fragmentation and capacitation status), scrotal circumference, expected predicted difference (EPD) for milk yield and fertility. Sahiwal bulls included in present study were 60 bulls used in breeding programme as well as 50 young bulls yet to be included in breeding programme. All the Sahiwal bulls were found to be polymorphic for CRISP2 gene (AA, AG and GG) present within exon 7 to the position 589 of CRISP2 mRNA by using PCR-SSCP and Sequencing. Semen analysis were done on 60 breeding bulls frozen semen doses pertaining to four season (winter, summer, rainy and autumn). The scrotal circumference was measured from existing Sahiwal breeding bulls in the herd (n=47). The effect of non-genetic factors on reproduction traits were studied by least-squares technique and the significant difference of means between subclasses of season, period, parity and age group were tested. The data were adjusted for the significant non-genetic factors to remove the differential environmental effects. The adjusted data were used to generate traits like Waiting Period (WP), Pregnancy Rate (PR), Expected Predicted Difference (EPD) of fertility, respectively. Genetic and phenotypic parameters of reproduction traits were estimated. The overall least-squares means of Age at First Calving (AFC), Service Period (SP) and WP were estimated as 36.69 ± 0.18 months, 120.47 ± 8.98 days and 79.78 ± 3.09 days respectively. Season and period of birth had significant effect (p < 0.01) on AFC. AFC was highest during autumn season of birth followed by summer, winter and rainy. Season and period of calving had significant effect (p < 0.01) on SP and WP of sahiwal cows. The WP for Sahiwal cows was standardized based on four developed predicted model for pregnancy rate 42, 63, 84 and 105 days using all lactation records. The WP for Sahiwal cows were standardized as 42 days. A selection criterion was developed for Sahiwal breeding bulls and young Sahiwal bulls on the basis of EPD of fertility. The genotype has significant effect on expected predicted difference of fertility and some semen parameters like post thaw motility and HOST. AA Genotype of CRISP2 gene revealed better EPD for fertility than EPD of milk yield. AA genotype of CRISP2 gene has higher scrotal circumference than other genotype. For young Sahiwal bulls only AA genotypes were present with similar patterns. So on the basis of association of genotype with seminal traits, EPD of milk yield and EPD for fertility status, AA and AG genotype of CRISP2 gene was better for higher fertility in Sahiwal bulls.

Keywords: expected predicted difference, fertility, sahiwal, waiting period

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Authors: Turin Datta, Kisor K. Sahu

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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

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Authors: Neeraj Vij, Amber Brennan, Jenni Winters, Hadi Salehi, Hamy Temkit, Emily Andrisevic, Mohan V. Belthur

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Idiopathic clubfoot is a common lower extremity deformity with an incidence of 1:500. The Ponseti Method is well known as the gold standard of treatment. However, there is limited functional data demonstrating correction of the clubfoot after treatment with the Ponseti method. The purpose of this study was to study the clinical and functional outcomes after the Ponseti method with the Clubfoot Disease-Specific Instrument (CDS) and pedobarography. This IRB-approved prospective study included patients aged 3-18 who were treated for idiopathic clubfoot with the Ponseti method between January 2008 and December 2018. Age-matched controls were identified through siblings of clubfoot patients and other community members. Treatment details were collected through a chart review of the included patients. Laboratory assessment included a physical exam, gait analysis, and pedobarography. The Pediatric Outcomes Data Collection Instrument and the Clubfoot Disease-Specific Instrument were also obtained on clubfoot patients (CF). The Wilcoxson rank-sum test was used to study differences between the CF patients and the typically developing (TD) patients. Statistical significance was set at p < 0.05. There were a total of 37 enrolled patients in our study. 21 were priorly treated for CF and 16 were TD. 94% of the CF patients had bilateral involvement. The age at the start of treatment was 29 days, the average total number of casts was seven to eight, and the average total number of casts after Achilles tenotomy was one. The reoccurrence rate was 25%, tenotomy was required in 94% of patients, and ≥1 tenotomy was required in 25% of patients. There were no significant differences between step length, step width, stride length, force-time integral, maximum peak pressure, foot progression angles, stance phase time, single-limb support time, double limb support time, and gait cycle time between children treated with the Ponseti method and typically developing children. The average post-treatment Pirani and Dimeglio scores were 5.50±0.58 and 15.29±1.58, respectively. The average post-treatment PODCI subscores were: Upper Extremity: 90.28, Transfers: 94.6, Sports: 86.81, Pain: 86.20, Happiness: 89.52, Global: 88.6. The average post-treatment Clubfoot Disease-Specific Instrument scores subscores were: Satisfaction: 73.93, Function: 80.32, Overall: 78.41. The Ponseti Method has a very high success rate and remains to be the gold standard in the treatment of idiopathic clubfoot. Timely management leads to good outcomes and a low need for repeated Achilles tenotomy. Children treated with the Ponseti method demonstrate good functional outcomes as measured through pedobarography. Pedobarography may have clinical utility in studying congenital foot deformities. Objective measures for hours of brace wear could represent an improvement in clubfoot care.

Keywords: functional outcomes, pediatric deformity, patient-reported outcomes, talipes equinovarus

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

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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

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Authors: Jonathan Sahu, Jill Aylott

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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

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Authors: Jyoti Sahu, Vinay A. Juvekar

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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

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

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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

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

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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

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