Search results for: Ila Shukla

Authors: Lubna Azmi, Ila Shukla, Shyam Sundar Gupta, Padam Kant, C. V. Rao

Abstract:

To investigate the chemoprotective potential of NBRIQU16 chemotype isolated from Argyreia speciosa (Family: Convolvulaceae) on N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and NaCl-induced gastric carcinomas in Wistar rats. Forty-six male 6-week-old Wistar rats were divided into two groups. Thirty rats in group A were fed with a diet supplemented with 8 % NaCl for 20 weeks and simultaneously given N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) in drinking water at a concentration of 100 ug/ml for the first 17 weeks. After administration of the carcinogen, 200 and 400 mg/kg of NBRIQU16 were administered orally once a day throughout the study. From week 18, these rats were given normal water. From week 21, these rats were fed with a normal diet for 15 weeks. Group B containing 16 rats was fed standard diet for thirty-five days. It served as control. Ten rats from group A were sacrificed after 20 weeks. Scarification of remaining animals was conducted after 35 weeks. Entire stomach and some part of the duodenum were incised parallel to the greater curvature, and the samples were collected. After opening the stomach location and size of tumors were recorded. The number of tumors with their locations and sizes were recorded. Expression of survivin was examined by recording the Immunohistochemistry of the specimens. The treatment with NBRIQU16 significantly reduced the nodule incidence and nodule multiplicity in the rats after MNNG administration. Surviving expression in glandular stomachs of normal rats, of rats in middle induction period, in adenocarcinomas and NBRIQU16 treated tissues adjacent to tumor were 0, 42.0 %, 79.3%, and 36.4 %, respectively. Expression of survivin was significantly different as compared to the normal rats. Histological observations of stomach tissues too correlated with the biochemical observations.These finding powerfully supports that NBRIQU16 chemopreventive effect by suppressing the tumor burden and restoring the activities of gastric cancer marker enzymes on MNNG and NaCl-induced gastric carcinomas in Wistar rats.

Keywords: Argyreia speciosa, gastric carcinoma, immunochemistry, NBRIQU16

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Authors: Tulika Malviya, Ritesh Chandra Shukla, Praveen Kumar Tandon

Abstract:

Traditional methods of synthesis are hazardous for the environment and need nature friendly processes for the treatment of industrial effluents and contaminated water. Use of plant parts for the synthesis provides an efficient alternative method. In this paper, we report an ecofriendly and nonhazardous biobased method to prepare zerovalent iron nanoparticles (ZVINPs) using the liquor of commercially available tea. Tea liquor as the reducing agent has many advantages over other polymers. Unlike other polymers, the polyphenols present in tea extract are nontoxic and water soluble at room temperature. In addition, polyphenols can form complexes with metal ions and thereafter reduce the metals. Third, tea extract contains molecules bearing alcoholic functional groups that can be exploited for reduction as well as stabilization of the nanoparticles. Briefly, iron nanoparticles were prepared by adding 2.0 g of montmorillonite K10 (MMT K10) to 5.0 mL of 0.10 M solution of Fe(NO3)3 to which an equal volume of tea liquor was then added drop wise over 20 min with constant stirring. The color of the mixture changed from whitish yellow to black, indicating the formation of iron nanoparticles. The nanoparticles were adsorbed on montmorillonite K10, which is safe and aids in the separation of hazardous arsenic species simply by filtration. Particle sizes ranging from 59.08±7.81 nm were obtained which is confirmed by using different instrumental analyses like IR, XRD, SEM, and surface area studies. Removal of arsenic was done via batch adsorption method. Solutions of As(III) of different concentrations were prepared by diluting the stock solution of NaAsO2 with doubly distilled water. The required amount of in situ prepared ZVINPs supported on MMT K10 was added to a solution of desired strength of As (III). After the solution had been stirred for the preselected time, the solid mass was filtered. The amount of arsenic [in the form of As (V)] remaining in the filtrate was measured using ion chromatograph. Stirring of contaminated water with zerovalent iron nanoparticles supported on montmorillonite K10 for 30 min resulted in up to 99% removal of arsenic as As (III) from its solution at both high and low pH (2.75 and 11.1). It was also observed that, under similar conditions, montmorillonite K10 alone provided only <10% removal of As(III) from water. Adsorption at low pH with precipitation at higher pH has been proposed for As(III) removal.

Keywords: arsenic removal, montmorillonite K10, tea liquor, zerovalent iron nanoparticles

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Authors: Neetika Mahajan, Awanish Shukla

Abstract:

Entrepreneurship is key a driver to economic development. It opens opportunities for business startups and has potential to expand employment opportunities for many. Entrepreneurship gives ‘Purpose thriving’ approach towards society with new technologies and zeal to develop and compete in the market. There are many more advantages of entrepreneurship like freedom to scope of work and independence in setting own goals. Women contribute to nearly 50 percent of India’s population, constitute nearly 10 percent of a total number of entrepreneurs in India. Women are found to be better risk calculators, more ambitious and less prone to self-confidence. However, It is a hard fact that life has not been easy for women aspiring professional success. Gender parity is the biggest threat faced by female aspirant seeking new businesses. More challenges like socio-cultural barriers, insufficient financial assistance, etc. have been faced by the women of our country. To uplift the status of women in the society, a number of initiatives have been taken up by the Government of India. Initiatives like National Mission for Empowerment of Women by (Ministry of Women And Child Development) and SKILL INDIA aim to increase the technical skills and knowledge of women for tapping employment opportunities and self-confidence. Trade Related Entrepreneurship Assistance and Development (TREAD) Scheme and Mahila Coir Yojana are proposed by the Ministry of MSMEs aiming to facilitate employment opportunities for women and entrepreneurship development. This paper will aim to bring out the gaps and barriers, which are still resisting the potential women come upfront and start a new business irrespective of a number of initiatives being put by government of India. The aim is also to identify focus areas where further intervention is required and proposing suitable interventions. The methodology to take forward this research will include primary and secondary data collection from on ground survey to track various kind of challenges faced by aspirant women entrepreneurs. Insight will be put towards initiations by the government of India towards women empowerment and assistance to entrepreneurship. Scientific quantitative tools will be used to analyze collected information. The final output of the research shall focus on achieving the respective aims and objectives.

Keywords: women entrepreneurship, government programs and schemes, key challenges, economic development

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Authors: Sameer A. Pathan, Biswadev Mitra, Salman Mirza, Umais Momin, Zahoor Ahmed, Lubna G. Andraous, Dharmesh Shukla, Mohammed Y. Shariff, Magid M. Makki, Tinsy T. George, Saad S. Khan, Stephen H. Thomas, Peter A. Cameron

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Study objective: Emergency physician’s (EP) ability to identify hydronephrosis on point-of-care ultrasound (POCUS) has been assessed in the past using CT scan as the reference standard. We aimed to assess EP interpretation of POCUS to identify and grade the hydronephrosis in a direct comparison with the consensus-interpretation of POCUS by radiologists, and also to compare the EP and radiologist performance using CT scan as the criterion standard. Methods: Using data from a POCUS databank, a prospective interpretation study was conducted at an urban academic emergency department. All POCUS exams were performed on patients presenting with renal colic to the ED. Institutional approval was obtained for conducting this study. All the analyses were performed using Stata MP 14.0 (Stata Corp, College Station, Texas). Results: A total of 651 patients were included, with paired sets of renal POCUS video clips and the CT scan performed at the same ED visit. Hydronephrosis was reported in 69.6% of POCUS exams by radiologists and 72.7% of CT scans (p=0.22). The κ for consensus interpretation of POCUS between the radiologists to detect hydronephrosis was 0.77 (0.72 to 0.82) and weighted κ for grading the hydronephrosis was 0.82 (0.72 to 0.90), interpreted as good to very good. Using CT scan findings as the criterion standard, Eps had an overall sensitivity of 81.1% (95% CI: 79.6% to 82.5%), specificity of 59.4% (95% CI: 56.4% to 62.5%), PPV of 84.3% (95% CI: 82.9% to 85.7%), and NPV of 53.8% (95% CI: 50.8% to 56.7%); compared to radiologist sensitivity of 85.0% (95% CI: 82.5% to 87.2%), specificity of 79.7% (95% CI: 75.1% to 83.7%), PPV of 91.8% (95% CI: 89.8% to 93.5%), and NPV of 66.5% (95% CI: 61.8% to 71.0%). Testing for a report of moderate or high degree of hydronephrosis, specificity of EP was 94.6% (95% CI: 93.7% to 95.4%) and to 99.2% (95% CI: 98.9% to 99.5%) for identifying severe hydronephrosis alone. Conclusion: EP POCUS interpretations were comparable to the radiologists for identifying moderate to severe hydronephrosis using CT scan results as the criterion standard. Among patients with moderate or high pre-test probability of ureteric calculi, as calculated by the STONE-score, the presence of moderate to severe (+LR 6.3 and –LR 0.69) or severe hydronephrosis (+LR 54.4 and –LR 0.57) was highly diagnostic of the stone disease. Low dose CT is indicated in such patients for evaluation of stone size and location.

Keywords: renal colic, point-of-care, ultrasound, bedside, emergency physician

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Authors: Ashi Gautam, Isha Shukla, Akhil Seghal

Abstract:

Biosensors are analytical devices that use a biological sensing element to detect and measure a specific chemical substance or biomolecule in a sample. These devices are widely used in various fields, including medical diagnostics, environmental monitoring, and food analysis, due to their high specificity, sensitivity, and selectivity. In this research paper, a machine learning model is proposed for predicting the suitability of skin care products based on biosensor readings. The proposed model takes in features extracted from biosensor readings, such as biomarker concentration, skin hydration level, inflammation presence, sensitivity, and free radicals, and outputs the most appropriate skin care product for an individual. This model is trained on a dataset of biosensor readings and corresponding skin care product information. The model's performance is evaluated using several metrics, including accuracy, precision, recall, and F1 score. The aim of this research is to develop a personalised skin care product recommendation system using biosensor data. By leveraging the power of machine learning, the proposed model can accurately predict the most suitable skin care product for an individual based on their biosensor readings. This is particularly useful in the skin care industry, where personalised recommendations can lead to better outcomes for consumers. The developed model is based on supervised learning, which means that it is trained on a labeled dataset of biosensor readings and corresponding skin care product information. The model uses these labeled data to learn patterns and relationships between the biosensor readings and skin care products. Once trained, the model can predict the most suitable skin care product for an individual based on their biosensor readings. The results of this study show that the proposed machine learning model can accurately predict the most appropriate skin care product for an individual based on their biosensor readings. The evaluation metrics used in this study demonstrate the effectiveness of the model in predicting skin care products. This model has significant potential for practical use in the skin care industry for personalised skin care product recommendations. The proposed machine learning model for predicting the suitability of skin care products based on biosensor readings is a promising development in the skin care industry. The model's ability to accurately predict the most appropriate skin care product for an individual based on their biosensor readings can lead to better outcomes for consumers. Further research can be done to improve the model's accuracy and effectiveness.

Keywords: biosensors, data model, machine learning, skin care

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Authors: Krupali Mehta, Sandip Bhatt, Umesh Trivedi, Bhavesh Bharatiya, Mukesh Ranjan, Atindra D. Shukla

Abstract:

Owing to the dominating surface forces and large-scale surface interactions, the nano-scale particles face difficulties in getting suspended in various media. Magnetic nanoparticles of iron oxide offer a great deal of promise due to their ease of preparation, reasonable magnetic properties, low cost and environmental compatibility. We intend to modify the surface of magnetic Fe₂O₃ nanoparticles with selected surface modifying agents using simple and effective single-step chemical reactions in order to enhance dispersibility of magnetic nanoparticles in non-polar media. Magnetic particles were prepared by hydrolysis of Fe²⁺/Fe³⁺ chlorides and their subsequent oxidation in aqueous medium. The dried particles were then treated with Octadecyl quaternary ammonium silane (Terrasil™), stearic acid and gallic acid ester of stearyl alcohol in ethanol separately to yield S-2 to S-4 respectively. The untreated Fe₂O₃ was designated as S-1. The surface modified nanoparticles were then analysed with Dynamic Light Scattering (DLS), Fourier Transform Infrared spectroscopy (FTIR), X-Ray Diffraction (XRD), Thermogravimetric Gravimetric Analysis (TGA) and Scanning Electron Microscopy and Energy dispersive X-Ray analysis (SEM-EDAX). Characterization reveals the particle size averaging 20-50 nm with and without modification. However, the crystallite size in all cases remained ~7.0 nm with the diffractogram matching to Fe₂O₃ crystal structure. FT-IR suggested the presence of surfactants on nanoparticles’ surface, also confirmed by SEM-EDAX where mapping of elements proved their presence. TGA indicated the weight losses in S-2 to S-4 at 300°C onwards suggesting the presence of organic moiety. Hydrophobic character of modified surfaces was confirmed with contact angle analysis, all modified nanoparticles showed super hydrophobic behaviour with average contact angles ~129° for S-2, ~139.5° for S-3 and ~151° for S-4. This indicated that surface modified particles are super hydrophobic and they are easily dispersible in non-polar media. These modified particles could be ideal candidates to be suspended in oil-based fluids, polymer matrices, etc. We are pursuing elaborate suspension/sedimentation studies of these particles in various oils to establish this conjecture.

Keywords: iron nanoparticles, modification, hydrophobic, dispersion

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Authors: Sanjay Shukla

Abstract:

Excessive use of antibiotics is a main problem in the treatment of wounds and other chronic infections and antibiotic treatment is frequently non-curative, thus alternative treatment is necessary. Phage therapy is considered one of the most effective approaches to treat multi-drug resistant bacterial pathogens. Infections caused by Staphylococcus aureus are very efficiently controlled with phage cocktails, containing a different individual phages lysate infecting a majority of known pathogenic S. aureus strains. The aim of current study was to investigate the efficiency of a purified phage cocktail for prophylactic as well as therapeutic application in mouse model and in large animals with chronic septic infection of wounds. A total of 150 sewage samples were collected from various livestock farms. These samples were subjected for the isolation of bacteriophage by double agar layer method. A total of 27 sewage samples showed plaque formation by producing lytic activity against S. aureus in double agar overlay method out of 150 sewage samples. In TEM recovered isolates of bacteriophages showed hexagonal structure with tail fiber. In the bacteriophage (ØVS) had an icosahedral symmetry with the head size 52.20 nm in diameter and long tail of 109 nm. Head and tail were held together by connector and can be classified as a member of the Myoviridae family under the order of Caudovirale. Recovered bacteriophage had shown the antibacterial activity against the S. aureus in vitro. Cocktail (ØVS1, ØVS5, ØVS9 and ØVS 27) of phage lysate were tested to know in vivo antibacterial activity as well as the safety profile. Result of mice experiment indicated that the bacteriophage lysate was very safe, did not show any appearance of abscess formation which indicates its safety in living system. The mice were also prophylactically protected against S. aureus when administered with cocktail of bacteriophage lysate just before the administration of S. aureus which indicates that they are good prophylactic agent. The S. aureus inoculated mice were completely recovered by bacteriophage administration with 100% recovery which was very good as compere to conventional therapy. In present study ten chronic cases of wound were treated with phage lysate and follow up of these cases was done regularly up to ten days (at 0, 5 and 10 d). Result indicated that the six cases out of ten showed complete recovery of wounds within 10 d. The efficacy of bacteriophage therapy was found to be 60% which was very good as compared to the conventional antibiotic therapy in chronic septic wounds infections. Thus, the application of lytic phage in single dose proved to be innovative and effective therapy for treatment of septic chronic wounds.

Keywords: phage therapy, phage lysate, antimicrobial resistance, S. aureus

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Authors: Rashmi Shukla, Yamini Bhusan Tripathi

Abstract:

Diabetic nephropathy (DN) is characterized as diabetic kidney disease which involves many pathways e.g. hyperactivated protein kinase c (PKC), polyol pathway, excess production of advanced glycation end product (AGEs) & free radical accumulation etc. All of them results to hypoxia followed by apoptosis of podocytes, glomerulosclerosis, extracellular matrix (ECM) accumulation and fibrosis resulting to irreversible changes in kidney. This is continuously rising worldwide and there are not enough specific drugs, to retard its progress. Due to increasing side effects of allopathic drugs, interest in herbal remedies is growing. Earlier, we have reported that PTY-2 (a phytomedicine, derived from Pueraria tuberosa Linn.) inhibits the accumulation of extracellular matrix (ECM) through activation of MMP-9. Present study exhibited the therapeutic potential of Pueraria tuberosa in the prevention of podocytes apoptosis and modulation of nephrin expression in streptozotocin (STZ) induced DN rats. DN rats were produced by maintaining persistent hyperglycemia for 8 weeks by intra-peritoneal injection of 55 mg/kg streptozotocin (STZ). These rats were randomly divided in 2 groups, i.e. DN control, and DN+ water extract of Pueraria tuberosa (PTW). One group of age-matched normal rats served as non-diabetic control (group-1), The STZ induced DN rats (group-2) and DN+PTW treated rats (group-3). The PTW was orally administered (0.3g/kg) daily to group-2 rats and drug vector (1 ml of 10% tween 20) in control rats. The treatments were continued for 20 days and blood and urine samples were collected. Rats were then sacrificed to investigate the expression Bcl2, Bax and nephroprotective protein i.e. nephrin in kidney glomerulus. The effect of PTW was evaluated, we have found that the PTW significantly(p < .001) reversed the raised serum urea, serum creatinine, urine protein and improved the creatinine clearance in STZ induce diabetic nephropathy in rats and also significantly(p < .001) prevented the rise in urine albumin excretion. The Western blot analysis of kidney tissue homogenate showed increased expression of Bcl2 in PTW treated rats. The RT-PCR showed the increased expression and accumulation of nephrin mRNA. The confocal photomicrographs also supported the reduction of Bax and a simultaneous increase in Bcl2 and nephrin in glomerular podocytes. Hence, our finding suggests that the nephroprotective role of PTW is mediated via restoration of nephrin thus prevents the podocytes apoptosis and ameliorates diabetic nephropathy. The clinical trial of PTW would prove to be a potential food supplement/ drug of alternative medicine for patients with diabetic nephropathy in early stage.

Keywords: Pueraria tuberosa, diabetic nephropathy, anti-apoptosis, nephrin

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Authors: Sanjay Shukla, A. Nayak, R. K. Sharma, A. P. Singh, S. P. Tiwari

Abstract:

Excessive use of antibiotics is a major problem in the treatment of wounds and other chronic infections, and antibiotic treatment is frequently non-curative, thus alternative treatment is necessary. Phage therapy is considered one of the most promising approaches to treat multi-drug resistant bacterial pathogens. Infections caused by Staphylococcus aureus are very efficiently controlled with phage cocktails, containing a different individual phages lysate infecting a majority of known pathogenic S. aureus strains. The aim of the present study was to evaluate the efficacy of a purified phage cocktail for prophylactic as well as therapeutic application in mouse model and in large animals with chronic septic infection of wounds. A total of 150 sewage samples were collected from various livestock farms. These samples were subjected for the isolation of bacteriophage by the double agar layer method. A total of 27 sewage samples showed plaque formation by producing lytic activity against S. aureus in the double agar overlay method out of 150 sewage samples. In TEM, recovered isolates of bacteriophages showed hexagonal structure with tail fiber. In the bacteriophage (ØVS) had an icosahedral symmetry with the head size 52.20 nm in diameter and long tail of 109 nm. Head and tail were held together by connector and can be classified as a member of the Myoviridae family under the order of Caudovirale. Recovered bacteriophage had shown the antibacterial activity against the S. aureus in vitro. Cocktail (ØVS1, ØVS5, ØVS9, and ØVS 27) of phage lysate were tested to know in vivo antibacterial activity as well as the safety profile. Result of mice experiment indicated that the bacteriophage lysate were very safe, did not show any appearance of abscess formation, which indicates its safety in living system. The mice were also prophylactically protected against S. aureus when administered with cocktail of bacteriophage lysate just before the administration of S. aureuswhich indicates that they are good prophylactic agent. The S. aureusinoculated mice were completely recovered by bacteriophage administration with 100% recovery, which was very good as compere to conventional therapy. In the present study, ten chronic cases of the wound were treated with phage lysate, and follow up of these cases was done regularly up to ten days (at 0, 5, and 10 d). The result indicated that the six cases out of ten showed complete recovery of wounds within 10 d. The efficacy of bacteriophage therapy was found to be 60% which was very good as compared to the conventional antibiotic therapy in chronic septic wounds infections. Thus, the application of lytic phage in single dose proved to be innovative and effective therapy for the treatment of septic chronic wounds.

Keywords: phage therapy, S aureus, antimicrobial resistance, lytic phage, and bacteriophage

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Authors: Mayoorika Shukla, Pramila Jakhar, Tejendra Dixit, I. A. Palani, Vipul Singh

Abstract:

Biosensors are analytical devices with wide range of applications in biological, chemical, environmental and clinical analysis. It comprises of bio-recognition layer which has biomolecules (enzymes, antibodies, DNA, etc.) immobilized over it for detection of analyte and transducer which converts the biological signal into the electrical signal. The performance of biosensor primarily the depends on the bio-recognition layer and therefore it has to be chosen wisely. In this regard, nanostructures of metal oxides such as ZnO, SnO2, V2O5, and TiO2, etc. have been explored extensively as bio-recognition layer. Recently, ZnO has the attracted attention of researchers due to its unique properties like high iso-electric point, biocompatibility, stability, high electron mobility and high electron binding energy, etc. Although there have been many reports on usage of ZnO as bio-recognition layer but to the authors’ knowledge, none has ever observed correlation between optical properties like defect suppression and biosensing capability of the sensor. Here, ZnO nanorods (ZNR) have been synthesized by a low cost, simple and low-temperature hydrothermal growth process, over Platinum (Pt) coated glass substrate. The ZNR have been synthesized in two steps viz. initially a seed layer was coated over substrate (Pt coated glass) followed by immersion of it into nutrient solution of Zinc nitrate and Hexamethylenetetramine (HMTA) with in situ addition of KMnO4. The addition of KMnO4 was observed to have a profound effect over the growth rate anisotropy of ZnO nanostructures. Clustered and powdery growth of ZnO was observed without addition of KMnO4, although by addition of it during the growth, uniform and crystalline ZNR were found to be grown over the substrate. Moreover, the same has resulted in suppression of defects as observed by Normalized Photoluminescence (PL) spectra since KMnO4 is a strong oxidizing agent which provides an oxygen rich growth environment. Further, to explore the correlation between defect suppression and biosensing capability of the ZNR Glucose oxidase (Gox) was immobilized over it, using physical adsorption technique followed by drop casting of nafion. Here the main objective of the work was to analyze effect of defect suppression over biosensing capability, and therefore Gox has been chosen as model enzyme, and electrochemical amperometric glucose detection was performed. The incorporation of KMnO4 during growth has resulted in variation of optical and charge transfer properties of ZNR which in turn were observed to have deep impact on biosensor figure of merits. The sensitivity of biosensor was found to increase by 12-18 times, due to variations introduced by addition of KMnO4 during growth. The amperometric detection of glucose in continuously stirred buffer solution was performed. Interestingly, defect suppression has been observed to contribute towards the improvement of biosensor performance. The detailed mechanism of growth of ZNR along with the overall influence of defect suppression on the sensing capabilities of the resulting enzymatic electrochemical biosensor and different figure of merits of the biosensor (Glass/Pt/ZNR/Gox/Nafion) will be discussed during the conference.

Keywords: biosensors, defects, KMnO4, ZnO nanorods

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

Abstract:

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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