Search results for: Ashish Kapoor

Authors: Rishabh Nagpal, Riya Uchagaonkar, Venkata Naga Narasimha Ashish Mernedi, Ahmed Hambaba

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The rapid evolution of technology in the communication sector continually seeks to bridge the gap between different communities, notably between the deaf community and the hearing world. This project develops a comprehensive American Sign Language (ASL) recognition system, leveraging the advanced capabilities of convolutional neural networks (CNNs) and vision transformers (ViTs) to interpret and translate ASL in real-time. The primary objective of this system is to provide an effective communication tool that enables seamless interaction through accurate sign language interpretation. The architecture of the proposed system integrates dual networks -VGG16 for precise spatial feature extraction and vision transformers for contextual understanding of the sign language gestures. The system processes live input, extracting critical features through these sophisticated neural network models, and combines them to enhance gesture recognition accuracy. This integration facilitates a robust understanding of ASL by capturing detailed nuances and broader gesture dynamics. The system is evaluated through a series of tests that measure its efficiency and accuracy in real-world scenarios. Results indicate a high level of precision in recognizing diverse ASL signs, substantiating the potential of this technology in practical applications. Challenges such as enhancing the system’s ability to operate in varied environmental conditions and further expanding the dataset for training were identified and discussed. Future work will refine the model’s adaptability and incorporate haptic feedback to enhance the interactivity and richness of the user experience. This project demonstrates the feasibility of an advanced ASL recognition system and lays the groundwork for future innovations in assistive communication technologies.

Keywords: sign language, computer vision, vision transformer, VGG16, CNN

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Authors: Kaesha M. Freyaldenhoven

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Chicago is a city in the United States whose cultural identity is largely shaped by public art pieces. Quintessential public works created by internationally renown artists – such as Anish Kapoor’s Cloud Gate in Millennium Park and 'The Picasso' in Daley Plaza – have historically contributed to developing a shared sense of community. In 2017, the city implemented a policy titled 50x50 Neighborhood Arts Project under the Chicago Public Art Plan. The policy promotes investments in contemporary public art to elevate neighborhood cultural assets and create a sense of place. Exclusively community-based artists were commissioned to accomplish the mission of the policy. Administrators felt only local artists would be capable of capturing the true essence of a neighborhood through art. This paper discusses the relationship between the public art and the culture of its respective neighborhood through close examination of aesthetic formal properties and social significance. Research compares the role of international artists with the role of local artists in cultivating the identity of a city through site-specific artworks in Chicago. Methodology unites theoretical research on understanding art and its function in the public space with empirical research on Chicago-based works. Theoretical frameworks provide an art historical foundation to explore the manner in which physical properties convey meaning through the work itself and its placement in an urban setting. Empirical research that examines policy documentation and press announcements released by the Department of Cultural Affairs and Special Events investigates project selection processes pertaining to the artists and neighborhoods. Ethnographies and interviews of individuals from diverse social segments in contemporary Chicago society measure impacts of the works on respective populations. Findings demonstrate works created by local artists activate neighborhoods and inculcate a sense of pride among community residents. Works created by international artists garner widespread media attention that frames the city’s cultural identity across temporal and geographic zones. This research can be utilized to inform future cultural policies pertaining to the commission of public art.

Keywords: Chicago, cultural policy, public art, urban art

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Authors: Mahendran Ganesan, Sanjeet Kumar Verma, Zafar Iqbal, Ashish Chandran, Zakir Husain, Shama Afroz, Sana Shahid, Laiq Ur Rahman

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Highly efficient in vitro regeneration system has been developed for Swertia chirayita (Roxb. ex Fleming) H. Karst, a high prized traditional medicinal plant to treat numerous ailments such as liver disorders, malaria and diabetes and are reported to have a wide spectrum of pharmacological properties. Its medicinal usage is well-documented in Indian pharmaceutical codex, the British and the American pharmacopeias, and in different traditional medicine such as the Ayurveda, Unani and Siddha medical systems. Nodal explants were cultured on MS medium supplemented with various phytohormones for multiple shoot induction. The nodal segments failed to respond in growth regulator free medium. All the concentrations of BAP, Kin and TDZ facilitated shoot bud break and multiple shoot induction. Among the various cytokinins tested, BAP was found to be more effective with respect to initiation and subsequent development of shoots. Of the various concentrations BAP tested, BAP at 4.0 mg/L showed the higher average number of shoot regeneration (10.80 shoots per explant). Kin at 4 mg/L and TDZ at 4 mg/L induced 5.70 and 04.5+0 shoots per explant, respectively. Further increase in concentration did not favour an increase in the number of shoots. However, these shoots failed to elongate further. Hence, addition of GA₃ (1 mg/L) was added to the above medium. This treatment resulted in the elongation of shoots (2.50 cm) and a further increase in the number of microshoots (34.20 shoots/explant). Roots were also induced in the same medium containing BAP (4 mg/L) + GA₃ (1 mg/L) + NAA (0.5 mg/L). In vitro derived plantlets with well-developed roots were transferred to the potting media containing garden soil: sand: vermicompost (2:1:1). Plantlets were covered with a polyethylene bag and irrigated with water. The pots were maintained at 25 ± 2ºC, and then the polyethylene cover was gradually loosened, thus dropping the humidity (65–70%). This procedure subsequently resulted in in vitro hardening of the plantlet.

Keywords: micropropagation, nodal explant, plant growth regulators, Swertia chirayita

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Authors: Ujala, Diksha Sharma, Mahinder Partap, Ashish R. Warghat, Bhavya Bhargava

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In soil-less agriculture, hydroponic is considered a potential farming system for the production of uniform quality plant material in significantly less time. Therefore, for the first time, the current investigation corroborates the effect of different cultivation conditions (open-field, poly-house, and hydroponic) on morpho-physiological traits, phenolic content, and essential oil components analysis in three flower color variants (yellow, scarlet red, and orange) of Tagetes patula. The results revealed that the maximum plant height, number of secondary branches, number of flowers, photosynthesis, stomatal conductance, and transpiration rate were observed under the hydroponic system as compared to other conditions. However, the maximum content of gallic acid (0.82 mg/g DW), syringic acid (3.98 mg/g DW), epicatechin (0.48 mg/g DW), p-coumaric acid (7.28 mg/g DW), protocatechuic acid (0.59 mg/g DW), ferulic acid (2.58 mg/g DW), and luteolin (8.24 mg/g DW) were quantified maximally under open-field conditions. However, under hydroponic conditions, the higher content of vanillic acid (0.43 mg/g DW), caffeic acid (0.49 mg/g DW), and quercetin (0.92 mg/g DW) were quantified. Moreover, a total of nineteen volatile components were identified in the essential oil of different flower color variants of T. patula cultivated under different conditions. The major reported volatile components in essential oil were (-)-caryophyllene oxide, trans-β-caryophyllene, trans-geraniol, 3 methyl-benzyl alcohol, and 2,2’:5’,2”-terthiophene. It has also been observed that the volatile component percentage range in all variants was observed in open-field (70.85 % to 90.54 %), poly-house (59.03 % to 77.93 %), and hydroponic (68.78 % to 89.41 %). In conclusion, the research highlighted that morpho-physiological performance with flower production was enhanced in the hydroponic system. However, phenolic content and volatile components were maximally observed in open-field conditions. However, significant results have been reported under hydroponic conditions in all studied parameters, so it could be a potential strategy for quality biomass production in T. patula.

Keywords: Tagetes patula, cultivation conditions, hydroponic, morpho-physiology

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Authors: Swati Srivastava, Ashish Kumar Upadhyay

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Background: According to United Nation Children’s Fund, it has been estimated that worldwide about 165 million children were stunted in 2012 and India alone accounts for 38% of global burden of stunting. In terms of incidence, India is home of more than 60 million stunted children worldwide. Our study aims to examine the effect of pregnancy intention and maternal postnatal depressive symptoms on early childhood stunting in India. We hypothesized that effect of pregnancy intention and postnatal maternal depressive symptoms were mediated by social support. Methods: We used data from first wave of Young Lives Study India. Out of 2011 children recruited in original cohort, 1833 children had complete information on pregnancy intention, maternal depression and other variables. A series of multivariate logistic regression model were used to examine the effect of pregnancy intention and postnatal depressive symptoms on early childhood stunting. Results: Bivariate result indicates that a higher percent of children born after unintended pregnancy (40%) were stunted than children of intended pregnancy (26%). Likewise, proportion of stunted children was also higher among women of high postnatal depressive symptoms (35%) than low level of depression (24%). Results of multivariate logistic regression model indicate that children born after unintended pregnancy were significantly more likely to be stunted than children born after intended pregnancy (Coefficient: 1.70, CI: 1.17, 2.48). Likewise, early childhood stunting was also associated with maternal postnatal depressive symptoms among women (Coefficient: 1.48, CI: 1.16, 1.88). The effect of pregnancy intention and postnatal depressive symptoms on early childhood stunting remains unchanged after controlling for social support and other variables. Conclusions: The findings of this study provide conclusive evidence regarding consequences of pregnancy intention and postnatal depressive symptoms on early childhood stunting in India. Therefore, there is need to identify the women with unintended pregnancy and incorporate the promotion of mental health into their national reproductive and child health programme.

Keywords: pregnancy intention, postnatal depressive symptoms, social support, childhood stunting, young lives study, India

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Authors: A. Venkatakrishnan, M. Juneja, S. Kapoor

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Background: Gastrointestinal dysfunction is an emerging co morbidity seen in autism and may further strengthen the association between autism and celiac disease. This is supported by increased rates (22-70%) of gastrointestinal symptoms like diarrhea, constipation, abdominal discomfort/pain, and gastrointestinal inflammation in children with the etiology of autism is still elusive. In addition to genetic factors, environmental factors such as toxin exposure, intrauterine exposure to certain teratogenic drugs, are being proposed as possible contributing factors in the etiology of Autism Spectrum Disorders (ASD) in cognizance with reports of increased gut permeability and high rates of gastrointestinal symptoms noted in children with ASD, celiac disease has also been proposed as a possible etiological factor. Despite insufficient evidence regarding the benefit of restricted diets in Autism, GFD has been promoted as an alternative treatment for ASD. This study attempts to discern any correlation between ASD and celiac disease. Objective: This cross sectional study aims to determine the proportion of celiac disease in children with ASD. Methods: Study included 155 participants aged 2-12 yrs, diagnosed as ASD as per DSM-5 attending the child development center at a tertiary care hospital in Northern India. Those on gluten free diet or having other autoimmune conditions were excluded. A detailed Performa was filled which included sociodemographic details, history of gastrointestinal symptoms, anthropometry, systemic examination, and pertinent psychological testing was done using was assessed using Developmental Profile-3(DP-3) for Developmental Quotient, Childhood Autism Rating Scale-2 (CARS-2) for severity of ASD, Vineland Adaptive Behavior Scales (VABS) for adaptive behavior, Child Behavior Checklist (CBCL) for behavioral problems and BAMBI (Brief Autism Mealtime Behavior Scales) for feeding problems. Screening for celiac was done by TTG-IgA levels, and total serum IgA levels were measured to exclude IgA deficiency. Those with positive screen were further planned for HLA typing and endoscopic biopsy. Results: A total of 155 cases were included, out of which 5 had low IgA levels and were hence excluded from the study. The rest 150 children had TTG levels below the ULN and normal total serum IgA level. History of Gastrointestinal symptoms was present in 51 (34%) cases abdominal pain was the most frequent complaint (16.6%), followed by constipation (12.6%). Diarrhea was seen in 8 %. Gastrointestinal symptoms were significantly more common in children with ASD above 5 yrs (p-value 0.006) and those who were verbal (p = 0.000). There was no significant association between socio-demographic factors, anthropometric data, or severity of autism with gastrointestinal symptoms. Conclusion: None of the150 patients with ASD had raised TTG levels; hence no association was found between ASD and celiac disease. There is no justification for routine screening for celiac disease in children with ASD. Further studies are warranted to evaluate association of Non Celiac Gluten Sensitivity with ASD and any role of gluten-free diet in such patients.

Keywords: autism, celiac, gastrointestinal, gluten

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Authors: Ashish Trivedi, Amol Singh

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In the recent years, the magnitude and frequency of disasters have increased at an alarming rate. Every year, more than 400 natural disasters affect global population. A large-scale disaster leads to destruction or damage to houses, thereby rendering a notable number of residents homeless. Since humanitarian response and recovery process takes considerable time, temporary establishments are arranged in order to provide shelter to affected population. These shelter areas are vital for an effective humanitarian relief; therefore, they must be strategically planned. Choosing the locations of temporary shelter areas for accommodating homeless people is critical to the quality of humanitarian assistance provided after a large-scale emergency. There has been extensive research on the facility location problem both in theory and in application. In order to deliver sufficient relief aid within a relatively short timeframe, humanitarian relief organisations pre-position warehouses at strategic locations. However, such approaches have received limited attention from the perspective of providing shelters to disaster-affected people. In present research work, this aspect of humanitarian logistics is considered. The present work proposes a hybrid decision support model to determine relative preference of potential shelter locations by assessing them based on key subjective criteria. Initially, the factors that are kept in mind while locating potential areas for establishing temporary shelters are identified by reviewing extant literature and through consultation from a panel of disaster management experts. In order to determine relative importance of individual criteria by taking into account subjectivity of judgements, a hybrid approach of fuzzy sets and Analytic Hierarchy Process (AHP) was adopted. Further, Technique for order preference by similarity to ideal solution (TOPSIS) was applied on an illustrative data set to evaluate potential locations for establishing temporary shelter areas for homeless people in a disaster scenario. The contribution of this work is to propose a range of possible shelter locations for a humanitarian relief organization, using a robust multi criteria decision support framework.

Keywords: AHP, disaster preparedness, fuzzy set theory, humanitarian logistics, TOPSIS, temporary shelters

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Authors: Ashish Sinha, Pushpend Agrawal

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Background: Patients with undiagnosed pulmonary TB predominantly act as reservoirs for its transmission through 10-15 secondary infections in the next 1-5 Yrs. Delays in the diagnosis and treatment may worsen the disease with increase the risk of death. Factors responsible for such delays by tracking patient pathways to treatment may help in planning better interventions. The provision of ‘free diagnosis and treatment’ forms the cornerstone of the National Tuberculosis Elimination Programme (NTEP). OOPE is defined as the money spent by the patient during TB care other than public health facilities. Free TB care at all health facilities could reduce out-of-pocket expenses to the minimum possible levels. Material and Methods: This cross-sectional study was conducted among randomly selected 252 TB patients from Nov – Oct 2022 by taking in-depth interviews following informed verbal consent. We documented their journey from initial symptoms until they reached the public health facility, along with their ‘out-of-pocket expenditure’ (OOPE) pertaining to TB care. Results: Total treatment delay was 91±72 days on average (median: 77days, IQR: 45-104 days), while the isolated patient delay was 31±45 days (median: 15 days, IQR: 0 days to 43 days); diagnostic delay; 57±60 days (median: 42days, IQR 14-78 days), treatment delay 19 ± 18 days (median: 15days, IQR: 11-19 days). A patient delay (> 30 days) was significantly associated with ignorance about classic symptoms of pulmonary TB, adoption of self-medication, illiteracy, and middle and lower social class. Diagnostic delay was significantly higher among those who contacted private health facilities, were unaware of signs and symptoms, had >2 consultations, and not getting an appropriate referral for TB care. Most (97%) of the study participants interviewed claimed to have incurred some expenditure.Median total expenses were 6155(IQR: 2625-15175) rupees. More than half 141 (56%) of the study participants had expenses >5000 rupees. Median transport expenses were 525(IQR: 200-1012) rupees; Median consultation expenses were 700(IQR: 200-1600) rupees; Median investigation expenses were 1000(IQR: 0-3025) rupees and the Median medicine expenses were 3350(IQR: 1300-7525).OOPE for consultation, investigation, and medicine was observed to be significantly higher among patients who ignored classical signs& symptoms of TB, repeated visits to private health facilities, and due to self-medication practices. Transport expenses and delays in seeking care at facilities were observed to have an upward trend with OOP Expenses (r =1). Conclusion: Delay in TB care due to low awareness about signs and symptoms of TB and poor seeking care, lack of proper consultation, and appropriate referrals reported by the study subjects indicate the areas which need proper attention by the program managers. Despite a centrally sponsored programme, the financial burden on TB patients is still in the unacceptable range. OOPE could be reduced as low as possible by addressing the responsible factors linked to it.

Keywords: patient pathway, delay, pulmonary tuberculosis, out of pocket expenses

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Authors: Ashish Thakur

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This technical paper was written in view of focusing the biomaterials and its various applications in modern industries. Author tires to elaborate not only the medical, infect plenty of application in other industries. The scope of the research area covers the wide range of physical, biological and chemical sciences that underpin the design of biomaterials and the clinical disciplines in which they are used. A biomaterial is now defined as a substance that has been engineered to take a form which, alone or as part of a complex system, is used to direct, by control of interactions with components of living systems, the course of any therapeutic or diagnostic procedure. Biomaterials are invariably in contact with living tissues. Thus, interactions between the surface of a synthetic material and biological environment must be well understood. This paper reviews the benefits and challenges associated with surface modification of the metals in biomedical applications. The paper also elaborates how the surface characteristics of metallic biomaterials, such as surface chemistry, topography, surface charge, and wettability, influence the protein adsorption and subsequent cell behavior in terms of adhesion, proliferation, and differentiation at the biomaterial–tissue interface. The chapter also highlights various techniques required for surface modification and coating of metallic biomaterials, including physicochemical and biochemical surface treatments and calcium phosphate and oxide coatings. In this review, the attention is focused on the biomaterial-associated infections, from which the need for anti-infective biomaterials originates. Biomaterial-associated infections differ markedly for epidemiology, aetiology and severity, depending mainly on the anatomic site, on the time of biomaterial application, and on the depth of the tissues harbouring the prosthesis. Here, the diversity and complexity of the different scenarios where medical devices are currently utilised are explored, providing an overview of the emblematic applicative fields and of the requirements for anti-infective biomaterials. In addition to this, chapter introduces nanomedicine and the use of both natural and synthetic polymeric biomaterials, focuses on specific current polymeric nanomedicine applications and research, and concludes with the challenges of nanomedicine research. Infection is currently regarded as the most severe and devastating complication associated to the use of biomaterials. Osteoporosis is a worldwide disease with a very high prevalence in humans older than 50. The main clinical consequences are bone fractures, which often lead to patient disability or even death. A number of commercial biomaterials are currently used to treat osteoporotic bone fractures, but most of these have not been specifically designed for that purpose. Many drug- or cell-loaded biomaterials have been proposed in research laboratories, but very few have received approval for commercial use. Polymeric nanomaterial-based therapeutics plays a key role in the field of medicine in treatment areas such as drug delivery, tissue engineering, cancer, diabetes, and neurodegenerative diseases. Advantages in the use of polymers over other materials for nanomedicine include increased functionality, design flexibility, improved processability, and, in some cases, biocompatibility.

Keywords: nanomedicine, tissue, infections, biomaterials

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Authors: Md. Samsuddin Ansari, Ashish Arora

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Every year 10 million people fall ill with one of the oldest diseases known as tuberculosis, caused by Mycobacterium tuberculosis. The success of M. tuberculosis as a pathogen is because of its ability to persist in host tissues. Multidrug resistance (MDR) mycobacteria cases increase every day, which is associated with efflux pumps controlled at the level of transcription. The transcription regulators of MDR transporters in bacteria belong to one of the following four regulatory protein families: AraC, MarR, MerR, and TetR. Phenolic acid decarboxylase repressor (PadR), like a family of transcription regulators, is closely related to the MarR family. Phenolic acid decarboxylase repressor (PadR) was first identified as a transcription factor involved in the regulation of phenolic acid stress response in various microorganisms (including Mycobacterium tuberculosis H37Rv). Recently research has shown that the PadR family transcription factors are global, multifunction transcription regulators. Rv0047c is a PadR subfamily-1 protein. We are exploring the biophysical and structural characterization of Rv0047c. The Rv0047 gene was amplified by PCR using the primers containing EcoRI and HindIII restriction enzyme sites cloned in pET-NH6 vector and overexpressed in DH5α and BL21 (λDE3) cells of E. coli following purification with Ni2+-NTA column and size exclusion chromatography. We did DSC to know the thermal stability; the Tm (transition temperature) of protein is 55.29ºC, and ΔH (enthalpy change) of 6.92 kcal/mol. Circular dichroism to know the secondary structure and conformation and fluorescence spectroscopy for tertiary structure study of protein. To understand the effect of pH on the structure, function, and stability of Rv0047c we employed spectroscopy techniques such as circular dichroism, fluorescence, and absorbance measurements in a wide range of pH (from pH-2.0 to pH-12). At low and high pH, it shows drastic changes in the secondary and tertiary structure of the protein. EMSA studies showed the specific binding of Rv0047c with its own 30-bp promoter region. To determine the effect of complex formation on the secondary structure of Rv0047c, we examined the CD spectra of the complex of Rv0047c with promoter DNA of rv0047. The functional role of Rv0047c was characterized by over-expressing the Rv0047c gene under the control of hsp60 promoter in Mycobacterium tuberculosis H37Rv. We have predicted the three-dimensional structure of Rv0047c using the Swiss Model and Modeller, with validity checked by the Ramachandra plot. We did molecular docking of Rv0047c with dnaA, through PatchDock following refinement through FireDock. Through this, it is possible to easily identify the binding hot-stop of the receptor molecule with that of the ligand, the nature of the interface itself, and the conformational change undergone by the protein pattern. We are using X-crystallography to unravel the structure of Rv0047c. Overall the studies show that Rv0047c may have transcription regulation along with providing an insight into the activity of Rv0047c in the pH range of subcellular environment and helps to understand the protein-protein interaction, a novel target to kill dormant bacteria and potential strategy for tuberculosis control.

Keywords: mycobacterium tuberculosis, phenolic acid decarboxylase repressor, Rv0047c, Circular dichroism, fluorescence spectroscopy, docking, protein-protein interaction

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Authors: Ashish Kumar Dwivedi

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Uttar Pradesh is largest part of Indian Federal system encapsulating twenty two million populations and has huge cultural, economic and religious diversity. The newly elected 18 months old state leadership of Uttar Pradesh has envisaged and initiated various proactive strides for the public grievance redressal and inclusive development schemes for all the sections of population from its very day of assumption of the office by Hon’ble Chief Minster Shri Yogi Adtiyanath. These initiatives also include Departmental responses via social media handles as Twitter, Facebook Page, and Web interaction. In the same course, every department of state government has been guided for the correct usage of verified social media handle separately and in co-ordination with other departments. These guidelines included making new WhatsApp groups to connect technocrats and politicians to communicate on common platform. Minister for Department of Infrastructure and Industrial Development, Shri Satish Mahana is a very popular leader and very intuitive statesman, has thousands of followers on social media and his accounts receive almost three hundred individually mentioned notifications from the various parts of Uttar Pradesh. These notifications primarily include problems related to livelihood and grievances concerned to department. To address these communications, a body of five experts has been set who are actively responding on various levels and increase bureaucratic engagements with marginalized sections of society. With reference to above background, this piece of research was conducted to analyze, categorize and derive effective implementation of public policies via social media platforms. This act of responsiveness has brought positive change in the mindset of population for the government, which was missed earlier. Department of Industrial Development in the Government is also inclined to attract investors aiming to become first trillion-dollar economy of India henceforth department also organized two major successful events in last one year. These events were also frame worked on social media platform to update 2.5 million population of state who is actively using social media in many ways. To analyze change scientifically, this study has been conducted and big data has been collected from October 2017 to September 2018 from the departmental social media handles as Twitter, Facebook, and emails. For this data, a statistical study has been conducted to analyze sentiments and expectations, specific and common requirement of communities, nature of grievances and their effective elucidation fitted into government policies. The control sample has also been taken from previous government activities to analyze the change. The statistical study used tools such as correlation study and principal component analysis. Also in this research communication, the modus operandi of grievance redressal, proliferation of government policies, connections to their beneficiaries and quick response procedure will be discussed.

Keywords: correlation study, principal component analysis, bureaucratic engagements, social media

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Authors: Priyanka Jain, Ashish K. Sharma, Esha Shukla, K. J. Mukherjee

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We propose a paradigm shift in our approach to design improved platforms for recombinant protein production, by addressing system level issues rather than the individual steps associated with recombinant protein synthesis like transcription, translation, etc. We demonstrate that by controlling and modulating the cellular stress response (CSR), which is responsible for feedback control of protein synthesis, we can generate hyper-producing strains. We did transcriptomic profiling of post-induction cultures, expressing different types of protein, to analyze the nature of this cellular stress response. We found significant down-regulation of substrate utilization, translation, and energy metabolism genes due to generation CSR inside the host cell. However, transcription profiling has also shown that many genes are up-regulated post induction and their role in modulating the CSR is unclear. We hypothesized that these up-regulated genes trigger signaling pathways, generating the CSR and concomitantly reduce the recombinant protein yield. To test this hypothesis, we knocked out the up-regulated genes, which did not have any downstream regulatees, and analyzed their impact on cellular health and recombinant protein expression. Two model proteins i.e., GFP and L-Asparaginase were chosen for this analysis. We observed a significant improvement in expression levels, with some knock-outs showing more than 7-fold higher expression compared to control. The 10 best single knock-outs were chosen to make 45 combinations of all possible double knock-outs. A further increase in expression was observed in some of these double knock- outs with GFP levels being highest in a double knock-out ΔyhbC + ΔelaA. However, for L-Asparaginase which is a secretory protein, the best results were obtained using a combination of ΔelaA+ΔcysW knock-outs. We then tested all the knock outs for their ability to enhance the expression of a 'difficult-to-express' protein. The Rubella virus E1 protein was chosen and tagged with sfGFP at the C-terminal using a linker peptide for easy online monitoring of expression of this fusion protein. Interestingly, the highest increase in Rubella-sGFP levels was obtained in the same double knock-out ΔelaA + ΔcysW (5.6 fold increase in expression yield compared to the control) which gave the highest expression for L-Asparaginase. However, for sfGFP alone, the ΔyhbC+ΔmarR knock-out gave the highest level of expression. These results indicate that there is a fair degree of commonality in the nature of the CSR generated by the induction of different proteins. Transcriptomic profiling of the double knock out showed that many genes associated with the translational machinery and energy biosynthesis did not get down-regulated post induction, unlike the control where these genes were significantly down-regulated. This confirmed our hypothesis of these genes playing an important role in the generation of the CSR and allowed us to design a strategy for making better expression hosts by simply knocking out key genes. This strategy is radically superior to the previous approach of individually up-regulating critical genes since it blocks the mounting of the CSR thus preventing the down-regulation of a very large number of genes responsible for sustaining the flux through the recombinant protein production pathway.

Keywords: cellular stress response, GFP, knock-outs, up-regulated genes

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