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N-acetyl-5-neuraminic acid (NeuAc) plays crucial role in improving the growth, brain development, brain health maintenance, and immunity enhancement of infants. Commercially, it is used in the production of antiviral drugs, infant milk formulas, cosmetics, dietary supplements, and pharmaceutical products. Because of the rapidly increasing demand, metabolic engineering approach has attracted increasing attention for NeuAc biosynthesis. However, knowledge of metabolite flux in biosynthetic pathways is one of the major challenges in the practice of metabolic engineering. So, an understanding of the flux of NeuAc is needed to determine its cellular level at real time. The analysis of the flux can only be performed using a tool that has the capacity to measure metabolite level in cells without affecting other metabolic processes. A Fluorescence Resonance Energy Transfer (FRET)-based genetically-encoded nanosensor has been generated in this study to monitor the level of NeuAc in prokaryotic and eukaryotic cells. Sialic acid periplasmic binding protein (SiaP) from Haemophilus influenzae was exploited as a sensory element for the generation of nanosensor. The enhanced cyan fluorescent protein (ECFP) and Venus were used as Fluroscence Resonance Energy Transfer (FRET) pair. The nanosensor, which was termed fluorescent indicator protein for sialic acid (FLIP-SA), was successfully transformed into, and expressed in Escherichia coli BL21 (DE3) cells. The expressed protein of the nanosensor was isolated and purified. The purified nanosensor protein was characterized to assess the affinity, specificity, and stability in the pH range. The developed nanosensor exhibited FRET change after addition to NeuAc. The developed nanosensor was highly specific, exhibited pH stability, and detected NeuAc levels in the nanomolar to milimolar range. FLIP-SA was successfully introduced in bacterial and yeast cells and reported the real-time intracellular levels of NeuAc non-invasively. The FLIP-SA is an excellent tool for the metabolic flux analysis of the NeuAc biosynthetic pathway and, thus, may help unravel the regulatory mechanism of the metabolic pathway of NeuAc. Furthermore, FLIP-SA can be used for the high-throughput screening of E. coli mutant libraries for varied NeuAc production levels.
Ruphi Naz; Mohammad K. Okla; Urooj Fatima; Mohd. Mohsin; Walid H. Soufan; Ibrahim A. Alaraidh; Mostafa A. Abdel-Maksoud; Altaf Ahmad. Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells. Frontiers in Nutrition 2021, 8, 1 .
AMA StyleRuphi Naz, Mohammad K. Okla, Urooj Fatima, Mohd. Mohsin, Walid H. Soufan, Ibrahim A. Alaraidh, Mostafa A. Abdel-Maksoud, Altaf Ahmad. Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells. Frontiers in Nutrition. 2021; 8 ():1.
Chicago/Turabian StyleRuphi Naz; Mohammad K. Okla; Urooj Fatima; Mohd. Mohsin; Walid H. Soufan; Ibrahim A. Alaraidh; Mostafa A. Abdel-Maksoud; Altaf Ahmad. 2021. "Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells." Frontiers in Nutrition 8, no. : 1.
Vitamin E plays an exemplary role in living organisms. α-Tocopherol is the most superior and active form of naturally occurring vitamin E that meets the requirements of human beings as it possesses the α-tocopherol transfer protein (α-TTP). α-Tocopherol deficiency can lead to severe anemia, certain cancers, several neurodegenerative and cardiovascular diseases, and most importantly male infertility. As a result of the depletion of its natural sources, researchers have tried to employ metabolic engineering to enhance α-tocopherol production to meet the human consumption demand. However, the metabolic engineering approach relies on the metabolic flux of a metabolite in its biosynthetic pathway. Analysis of the metabolic flux of a metabolite needs a method that can monitor the α-tocopherol level in living cells. This study was undertaken to construct a FRET (fluorescence resonance energy transfer)-based nanosensor for monitoring the α-tocopherol flux in prokaryotic and eukaryotic living cells. The human α-TTP was sandwiched between a pair of FRET fluorophores to construct the nanosensor, which was denoted as FLIP-α (the fluorescence indicator for α-tocopherol). FLIP-α showed excellence in monitoring the α-tocopherol flux with high specificity. The sensor was examined for its pH stability for physiological applications, where it shows no pH hindrance to its activity. The calculated affinity of this nanosensor was 100 μM. It monitored the real-time flux of α-tocopherol in bacterial and yeast cells, proving its biocompatibility in monitoring the α-tocopherol dynamics in living cells. Being noninvasive, FLIP-α provides high temporal and spatial resolutions, which holds an indispensable significance in bioimaging metabolic pathways that are highly compartmentalized.
Habiba Kausar; Ghazala Ambrin; Mohammad K. Okla; Saud A. Alamri; Walid H. Soufan; Eid I. Ibrahim; Mostafa A. Abdel-Maksoud; Altaf Ahmad. FRET-Based Genetically Encoded Nanosensor for Real-Time Monitoring of the Flux of α-Tocopherol in Living Cells. ACS Omega 2021, 6, 9020 -9027.
AMA StyleHabiba Kausar, Ghazala Ambrin, Mohammad K. Okla, Saud A. Alamri, Walid H. Soufan, Eid I. Ibrahim, Mostafa A. Abdel-Maksoud, Altaf Ahmad. FRET-Based Genetically Encoded Nanosensor for Real-Time Monitoring of the Flux of α-Tocopherol in Living Cells. ACS Omega. 2021; 6 (13):9020-9027.
Chicago/Turabian StyleHabiba Kausar; Ghazala Ambrin; Mohammad K. Okla; Saud A. Alamri; Walid H. Soufan; Eid I. Ibrahim; Mostafa A. Abdel-Maksoud; Altaf Ahmad. 2021. "FRET-Based Genetically Encoded Nanosensor for Real-Time Monitoring of the Flux of α-Tocopherol in Living Cells." ACS Omega 6, no. 13: 9020-9027.
Increased problems associated with side effects and bacterial resistance of chemical drugs has prompted the research focus on herbal medicines in the past few decades. In the present investigation, the antimicrobial activity of the various parts of Avicennia marina (AM), a mangrove plant, has been evaluated. The plants were collected from the Jazan area of the Kingdom of Saudi Arabia. Primary extracts of roots, stem, leaves, fruits, and seeds were made in ethanol and fractioned in ethanol, ethyl acetate, petroleum ether, chloroform, and water. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the extracts were determined against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. It has been observed that the chloroform extract of roots of the AM exhibited inhibitory effects against both S. aureus (MIC = 1.5 ± 0.03 mg/mL) and E. coli (MIC = 1.7 ± 0.01 mg/mL). The ethanolic extract of the AM roots has shown antibacterial activity against Pseudomonas aeruginosa (MIC = 10.8 ± 0.78 mg/mL), Bacillus subtilis (MIC = 6.1 ± 0.27 mg/mL), Staphylococcus aureus (MIC = 2.3 ± 0.08 mg/mL), and Escherichia coli (MIC = 6.3 ± 0.28 mg/mL). The leaf extract of the AM in ethyl acetate showed antibacterial activity against S. aureus and E. coli. Antifungal activity of these extracts was also investigated against Aspergillus fumigatus and Candida albicans. Ethanolic extract of roots and seeds of the AM has shown antifungal activity against Aspergillus fumigatus when applied individually. Ethanolic extract of the AM fruits has shown an inhibitory effect on the growth of Aspergillus fumigatus and Candida albicans. It is suggested that the plant extracts of AM have tremendous antimicrobial activity against a group of microbes, and this effect depends on both the plant part and the solvent used for extraction. Therefore, this plant can be considered to treat various diseases caused by antibiotic-resistant bacteria.
Mohammad Okla; Abdulrahman Alatar; Saud Al-Amri; Walid Soufan; Altaf Ahmad; Mostafa Abdel-Maksoud. Antibacterial and Antifungal Activity of the Extracts of Different Parts of Avicennia marina (Forssk.) Vierh. Plants 2021, 10, 252 .
AMA StyleMohammad Okla, Abdulrahman Alatar, Saud Al-Amri, Walid Soufan, Altaf Ahmad, Mostafa Abdel-Maksoud. Antibacterial and Antifungal Activity of the Extracts of Different Parts of Avicennia marina (Forssk.) Vierh. Plants. 2021; 10 (2):252.
Chicago/Turabian StyleMohammad Okla; Abdulrahman Alatar; Saud Al-Amri; Walid Soufan; Altaf Ahmad; Mostafa Abdel-Maksoud. 2021. "Antibacterial and Antifungal Activity of the Extracts of Different Parts of Avicennia marina (Forssk.) Vierh." Plants 10, no. 2: 252.
Hydrogen peroxide (H2O2) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H2O2 increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H2O2 also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H2O2 in environmental responses and its role in cellular signaling, there is a need to study the flux of H2O2 at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H2O2) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H2O2, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H2O2 demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H2O2 was measured in these live cells using the FLIP-H2O2 under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H2O2. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.
Amreen; Hayssam M. Ali; Mohammad Ahmad; Mohamed Z. M. Salem; Altaf Ahmad. Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells. Biology 2020, 9, 430 .
AMA StyleAmreen, Hayssam M. Ali, Mohammad Ahmad, Mohamed Z. M. Salem, Altaf Ahmad. Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells. Biology. 2020; 9 (12):430.
Chicago/Turabian StyleAmreen; Hayssam M. Ali; Mohammad Ahmad; Mohamed Z. M. Salem; Altaf Ahmad. 2020. "Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells." Biology 9, no. 12: 430.
Nitrate (NO3–) is a critical source of nitrogen (N) available to microorganisms and plants. Nitrate sensing activates signaling pathways in the plant system that impinges upon, developmental, molecular, metabolic, and physiological responses locally, and globally. To sustain, the high crop productivity and high nutritional value along with the sustainable environment, the study of rate-controlling steps of a metabolic network of N assimilation through fluxomics becomes an attractive strategy. To monitor the flux of nitrate, we developed a non-invasive genetically encoded fluorescence resonance energy transfer (FRET)-based tool named “FLIP-NT” that monitors the real-time uptake of nitrate in the living cells. The developed nanosensor is suitable for real-time monitoring of nitrate flux in living cells at subcellular compartments with high spatio-temporal resolution. The developed FLIP-NT nanosensor was not affected by the pH change and have specificity for nitrate with an affinity constant (Kd) of ∼5 μM. A series of affinity mutants have also been generated to expand the physiological detection range of the sensor protein with varying Kd values. It has been found that this sensor successfully detects the dynamics of nitrate fluctuations in bacteria and yeast, without the disruption of cellular organization. This FLIP-NT nanosensor could be a very important tool that will help us to advance the understanding of nitrate signaling.
Urooj Fatima; Fuad Ameen; Neha Soleja; Parvez Khan; Abobakr Almansob; Altaf Ahmad. A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells. ACS Omega 2020, 5, 30306 -30314.
AMA StyleUrooj Fatima, Fuad Ameen, Neha Soleja, Parvez Khan, Abobakr Almansob, Altaf Ahmad. A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells. ACS Omega. 2020; 5 (46):30306-30314.
Chicago/Turabian StyleUrooj Fatima; Fuad Ameen; Neha Soleja; Parvez Khan; Abobakr Almansob; Altaf Ahmad. 2020. "A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells." ACS Omega 5, no. 46: 30306-30314.
Nitrogen (N), applied in the form of a nitrogenous fertilizer, is one of the main inputs for agricultural production. Food production is closely associated with the application of N. However, the application of nitrogenous fertilizers to agricultural fields is associated with heavy production of nitrous oxide because agricultural crops can only utilize 30–40% of applied N, leaving behind unused 60–70% N in the environment. The global warming effect of this greenhouse gas is approximately 300 times more than of carbon dioxide. Under the present scenario of climate change, it is critical to maintain the natural balance between food production and environmental sustainability by targeting traits responsible for improving nitrogen-use-efficiency (NUE). Understanding of the molecular mechanisms behind the metabolic alterations due to nitrogen status needs to be addressed. Additionally, mineral nutrient deficiencies and their associated metabolic networks have not yet been studied well. Given this, the alterations in core metabolic pathways of low-N tolerant (LNT) and low-N sensitive (LNS) genotypes of maize under N-deficiency and their efficiency of recovering the changes upon resupplying N were investigated by us, using the GC–MS and LC–MS based metabolomic approach. Significant genotype-specific changes were noted in response to low-N. The N limitation affected the whole plant metabolism, most significantly the precursors of primary metabolic pathways. These precursors may act as important targets for improving the NUE. Limited availability of N reduced the levels of N-containing metabolites, organic acids and amino acids, but soluble sugars increased. Major variations were encountered in LNS, as compared to LNT. This study has revealed potential metabolic targets in response to the N status, which are indeed the prospective targets for crop improvement.
Arshid Hussain Ganie; Renu Pandey; M. Nagaraj Kumar; Viswanathan Chinnusamy; Muhammad Iqbal; Altaf Ahmad. Metabolite Profiling and Network Analysis Reveal Coordinated Changes in Low-N Tolerant and Low-N Sensitive Maize Genotypes under Nitrogen Deficiency and Restoration Conditions. Plants 2020, 9, 1459 .
AMA StyleArshid Hussain Ganie, Renu Pandey, M. Nagaraj Kumar, Viswanathan Chinnusamy, Muhammad Iqbal, Altaf Ahmad. Metabolite Profiling and Network Analysis Reveal Coordinated Changes in Low-N Tolerant and Low-N Sensitive Maize Genotypes under Nitrogen Deficiency and Restoration Conditions. Plants. 2020; 9 (11):1459.
Chicago/Turabian StyleArshid Hussain Ganie; Renu Pandey; M. Nagaraj Kumar; Viswanathan Chinnusamy; Muhammad Iqbal; Altaf Ahmad. 2020. "Metabolite Profiling and Network Analysis Reveal Coordinated Changes in Low-N Tolerant and Low-N Sensitive Maize Genotypes under Nitrogen Deficiency and Restoration Conditions." Plants 9, no. 11: 1459.
Nanobiotechnological improvements defined on the utilization of biological materials and principles have enormously partaken to revolutionize physical, chemical, and biological sciences. However, the exploration of plant nanobiotechnology is still in its outset. The search for novel tools to monitor plant biomolecules is an emerging issue for the nanobiotechnologists. Given this, a genetically encoded FRET-based nanobiosensor has been developed to monitor the popular plant cardiac glycoside – digoxin, which is used as the most common prescription drug for heart-related illnesses across the world. Digoxin is sourced from the leaves of the foxglove plant (Digitalis purpurea L.) and has a significant demand in the medical sector. Moreover, with the rising popularity of the herbal formulations in the global market, attention towards the authentication and quality control of the herbal drugs is important. Furthermore, digoxin has a very narrow therapeutic range, i.e., 0.6 nM - 2.6 nM. Therefore, strict monitoring of blood digoxin levels is necessary. Besides, previously used techniques for drug authentication and quantification of small-molecule drugs in blood samples are not the best choice available. The nanobiosensor is based on the FRET (Fluorescence Resonance Energy Transfer) mechanism, and it is constructed in such a way that it gives a changed FRET output in the presence of digoxin. Two fluorophores, enhanced cyan fluorescent protein (ECFP) and Venus, were attached on either end of the sensory domain - human nuclear receptor ROR-gamma (RORγt). The developed nanobiosensor was named as fluorescent indicator protein for digoxin, (FLIP-digoxin). The ligand binding affinity of FLIP-digoxin was calculated as 425 μM. Affinity mutants of the FLIP-digoxin were also generated to measure digoxin in wide concentration ranges. This sensor offers high-throughput qualitative analysis of digoxin in Digitalis preparations procured from local drug stores. It confirms the authenticity of the preparations through the detection of digoxin. The FLIP-1n was also able to monitor digoxin concentration in serum samples in lesser than 5 min. The nanobiosensor was found pH stable, digoxin-specific, non- interfered by the biological serum species and can perform high throughput screening of the Digitalis powder, infusion and tincture preparations.
Ghazala Ambrin; Habiba Kausar; Altaf Ahmad. Designing and construction of genetically encoded FRET-based nanosensor for qualitative analysis of digoxin. Journal of Biotechnology 2020, 323, 322 -330.
AMA StyleGhazala Ambrin, Habiba Kausar, Altaf Ahmad. Designing and construction of genetically encoded FRET-based nanosensor for qualitative analysis of digoxin. Journal of Biotechnology. 2020; 323 ():322-330.
Chicago/Turabian StyleGhazala Ambrin; Habiba Kausar; Altaf Ahmad. 2020. "Designing and construction of genetically encoded FRET-based nanosensor for qualitative analysis of digoxin." Journal of Biotechnology 323, no. : 322-330.
Phosphorus (P) deficiency is one of the major limiting factors for crop productivity. The yield of rice (Oryza sativa L.) is severely limited by phosphorus deficiency. An attempt has been made in this study to identify P deficiency responsive differentially expressed proteins of rice through analysis of leaf proteome of contrasting P-responsive rice cultivars under P deficiency conditions because genetic variability has been found in the rice cultivars for adaptive response to P deficiency and a controlled regulatory system is involved in the P deficiency adaptation response. Phosphorus-efficient (cv. Panvel) and P-inefficient (cv. Nagina 22) rice cultivars were hydroponically grown in the nutrient medium under control environmental conditions at low-P level (2.0 µM) and optimum-P level (320 µM) treatments. Expression patterns of the proteins of the leaves of both the cultivars were analyzed in 30-day-old plants. The identification of these proteins through mass spectrometry and MASCOT software (Matrix Science Inc., Boston, USA) revealed that these differentially expressed proteins were homologous to known functional proteins involved in energy metabolism, biosynthesis, photosynthesis, signaling, protein synthesis, protein folding, phospholipid metabolism, oxidative stress, transcription factors, and phosphorus metabolism. It has been observed that rice cultivars responded differently to low-P treatment through modification in protein expressions pattern to maintain the growth of the plants. Therefore, the expression patterns of proteins were different in both of the cultivars under low-P treatment. Higher potential of protein stability, stress tolerance, osmo-protection, and regulation of phosphorus uptake was observed in cv. Panvel than cv. Nagina 22. This study could help to unravel the complex regulatory process that is involved in adaptation to P deficiency in rice.
Aadil Yousuf Tantray; Hayssam M. Ali; Altaf Ahmad. Analysis of Proteomic Profile of Contrasting Phosphorus Responsive Rice Cultivars Grown under Phosphorus Deficiency. Agronomy 2020, 10, 1028 .
AMA StyleAadil Yousuf Tantray, Hayssam M. Ali, Altaf Ahmad. Analysis of Proteomic Profile of Contrasting Phosphorus Responsive Rice Cultivars Grown under Phosphorus Deficiency. Agronomy. 2020; 10 (7):1028.
Chicago/Turabian StyleAadil Yousuf Tantray; Hayssam M. Ali; Altaf Ahmad. 2020. "Analysis of Proteomic Profile of Contrasting Phosphorus Responsive Rice Cultivars Grown under Phosphorus Deficiency." Agronomy 10, no. 7: 1028.
Ajmalicine is one of the most popular antihypertensive drugs obtained from the root barks of Cathranthus roseus (L.) G. Don and Rauvolfia serpentine (L.) Benth. ex Kurz. It has also potential antimicrobial, cytotoxic, central depressant and antioxidant activities. As the demand for the alkaloid is significantly high, metabolic engineering approaches are being tried to increase its production in both homologous and heterologous systems. The metabolic engineering approach requires knowledge of the metabolic regulation of the alkaloid. For understanding the metabolic regulation, fluxomic analysis is important as it helps in understanding the flux of the alkaloid through the complicated metabolic pathway. The present study was conducted to analyse the flux analysis of the ajmalicine biosynthesis, using a genetically encoded Fluorescent Resonance Energy Transfer FRET-based nanosensor for ajmalicine (FLIP-Ajn). Here, we have silenced six important genes of terpenoid indole alkaloid (TIA), namely G10H, 10HGO, TDC, SLS, STR and SDG, through RNA-mediated gene silencing in different batches of C. roseus suspension cells, generating six silenced cell lines. Monitoring of the ajmalicine level was carried out using FLIP-Ajn in these silenced cell lines, with high spatial and temporal resolution. The study offers the rapid, high throughput real-time measurement of ajmalicine flux in response to the silenced TIA genes, thereby identifying the regulatory gene controlling the alkaloid flux in C. roseus suspension cells. We have reported that the STR gene encoding strictosidine synthase of the TIA pathway could be the regulatory gene of the ajmalicine biosynthesis.
Ghazala Ambrin; Hayssam M. Ali; Altaf Ahmad. Metabolic Regulation Analysis of Ajmalicine Biosynthesis Pathway in Catharanthus roseus (L.) G. Don Suspension Culture Using Nanosensor. Processes 2020, 8, 589 .
AMA StyleGhazala Ambrin, Hayssam M. Ali, Altaf Ahmad. Metabolic Regulation Analysis of Ajmalicine Biosynthesis Pathway in Catharanthus roseus (L.) G. Don Suspension Culture Using Nanosensor. Processes. 2020; 8 (5):589.
Chicago/Turabian StyleGhazala Ambrin; Hayssam M. Ali; Altaf Ahmad. 2020. "Metabolic Regulation Analysis of Ajmalicine Biosynthesis Pathway in Catharanthus roseus (L.) G. Don Suspension Culture Using Nanosensor." Processes 8, no. 5: 589.
Sulfur (S) is an essential element for all forms of life. It is involved in numerous essential processes because S is considered as the primary source of one of the essential amino acids, methionine, which plays an important role in biological events. For the control and regulation of sulfate in a metabolic network through fluxomics, a non-invasive tool is highly desirable that opens the door to monitor the level of the sulfate in real time and space in living cells without fractionation of the cells or tissue. Here, we engineered a FRET (fluorescence resonance energy transfer) based sensor for sulfate, which is genetically-encoded and named as FLIP-SP (Fluorescent indicator protein for sulfate). The FLIP-SP can measure the level of the sulfate in live cells. This sensor was constructed by the fusion of fluorescent proteins at the N- and C-terminus of sulfate binding protein (sbp). The FLIP-SP is highly specific to sulfate, and showed pH stability. Real-time monitoring of the level of sulfate in prokaryotic and eukaryotic cells showed sensor bio-compatibility with living cells. We expect that this sulfate sensor offers a valuable strategy in the understanding of the regulation of the flux of sulfate in the metabolic network.
Urooj Fatima; Mohammad K. Okla; Mohd. Mohsin; Ruphi Naz; Walid Soufan; Abdullah A. Al-Ghamdi; Altaf Ahmad. A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells. International Journal of Molecular Sciences 2020, 21, 2572 .
AMA StyleUrooj Fatima, Mohammad K. Okla, Mohd. Mohsin, Ruphi Naz, Walid Soufan, Abdullah A. Al-Ghamdi, Altaf Ahmad. A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells. International Journal of Molecular Sciences. 2020; 21 (7):2572.
Chicago/Turabian StyleUrooj Fatima; Mohammad K. Okla; Mohd. Mohsin; Ruphi Naz; Walid Soufan; Abdullah A. Al-Ghamdi; Altaf Ahmad. 2020. "A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells." International Journal of Molecular Sciences 21, no. 7: 2572.
(+)-Catechin is an important antioxidant of green tea (Camelia sinensis (L.) O. Kuntze). Catechin is known for its positive role in anticancerous activity, extracellular matrix degradation, cell death regulation, diabetes, and other related disorders. As a result of enormous interest in and great demand for catechin, its biosynthesis using metabolic engineering has become the subject of concentrated research with the aim of enhancing (+)-catechin production. Metabolic flux is an essential concept in the practice of metabolic engineering as it helps in the identification of the regulatory element of a biosynthetic pathway. In the present study, an attempt was made to analyze the metabolic flux of the (+)-catechin biosynthesis pathway in order to decipher the regulatory element of this pathway. Firstly, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor (FLIP-Cat, fluorescence indicator protein for (+)-catechin) was developed for real-time monitoring of (+)-catechin flux. In vitro characterization of the purified protein of the nanosensor showed that the nanosensor was pH stable and (+)-catechin specific. Its calculated Kd was 139 µM. The nanosensor also performed real-time monitoring of (+)-catechin in bacterial cells. In the second step of this study, an entire (+)-catechin biosynthesis pathway was constructed and expressed in E. coli in two sets of plasmid constructs: pET26b-PT7-rbs-PAL-PT7-rbs-4CL-PT7-rbs-CHS-PT7-rbs-CHI and pET26b-T7-rbs-F3H-PT7-rbs- DFR-PT7-rbs-LCR. The E. coli harboring the FLIP-Cat was transformed with these plasmid constructs. The metabolic flux analysis of (+)-catechin was carried out using the FLIP-Cat. The FLIP-Cat successfully monitored the flux of catechin after adding tyrosine, 4-coumaric acid, 4-coumaroyl CoA, naringenin chalcone, naringenin, dihydroquercetin, and leucocyanidin, individually, with the bacterial cells expressing the nanosensor as well as the genes of the (+)-catechin biosynthesis pathway. Dihydroflavonol reductase (DFR) was identified as the main regulatory element of the (+)-catechin biosynthesis pathway. Information about this regulatory element of the (+)-catechin biosynthesis pathway can be used for manipulating the (+)-catechin biosynthesis pathway using a metabolic engineering approach to enhance production of (+)-catechin.
Habiba Kausar; Ghazala Ambrin; Mohammad K. Okla; Walid Soufan; Abdullah A. Al-Ghamdi; Altaf Ahmad. Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor. Antioxidants 2020, 9, 288 .
AMA StyleHabiba Kausar, Ghazala Ambrin, Mohammad K. Okla, Walid Soufan, Abdullah A. Al-Ghamdi, Altaf Ahmad. Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor. Antioxidants. 2020; 9 (4):288.
Chicago/Turabian StyleHabiba Kausar; Ghazala Ambrin; Mohammad K. Okla; Walid Soufan; Abdullah A. Al-Ghamdi; Altaf Ahmad. 2020. "Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor." Antioxidants 9, no. 4: 288.
An efficient protocol of plant regeneration through indirect organogenesis in Viola serpens was developed in the present study. Culture of leaf explants on MS (Murashige and Skoog) medium supplemented with 2.0 mg/L 6-benzyladenine and 0.13 mg/L 2,4-dichloro phenoxy acetic acid. Adventitious shoot formation was observed when calli were transferred on to MS medium containing 0.5 mg/L α-naphthalene acetic acid and 2.25 mg/L kinetin, which showed the maximum 86% shoot regeneration frequency. The highest root frequency (80.92%) with the 5.6 roots per explant and 1.87 cm root length was observed on MS medium supplemented with 2 mg/L indole-3-butyric acid. The plantlets were transferred to the mixture of sand, coffee husk and soil in the ratio of 1:2:1 in a pot, and placed under 80% shade net for one month. It was then transferred to 30% shade net for another one month, prior to transplantation in the field. These plantlets successfully acclimatized under field conditions. A Sequence Characterized Amplified Region (SCAR) marker was also developed using a 1135 bp amplicon that was obtained from RAPD (Random Amplification of Polymorphic DNA) analysis of six accessions of V. serpens. Testing of several market samples of V. serpens using the SCAR marker revealed successful identification of the genuine samples of V. serpens. This study, therefore, provides a proficient in vitro propagation protocol of V. serpens using leaf explants and a SCAR marker for the authentic identification of V. serpens. This study will be helpful for conservation of authentic V. serpens.
Shipra Rani Jha; Ruphi Naz; Ambreen Asif; Mohammad K. Okla; Walid Soufan; Abdullah A. Al-Ghamdi; Altaf Ahmad. Development of an In Vitro Propagation Protocol and a Sequence Characterized Amplified Region (SCAR) Marker of Viola serpens Wall. ex Ging. Plants 2020, 9, 246 .
AMA StyleShipra Rani Jha, Ruphi Naz, Ambreen Asif, Mohammad K. Okla, Walid Soufan, Abdullah A. Al-Ghamdi, Altaf Ahmad. Development of an In Vitro Propagation Protocol and a Sequence Characterized Amplified Region (SCAR) Marker of Viola serpens Wall. ex Ging. Plants. 2020; 9 (2):246.
Chicago/Turabian StyleShipra Rani Jha; Ruphi Naz; Ambreen Asif; Mohammad K. Okla; Walid Soufan; Abdullah A. Al-Ghamdi; Altaf Ahmad. 2020. "Development of an In Vitro Propagation Protocol and a Sequence Characterized Amplified Region (SCAR) Marker of Viola serpens Wall. ex Ging." Plants 9, no. 2: 246.
Reduced glutathione (GSH) level inside the cell is a critical determinant for cell viability. The level of GSH varies across the cells, tissues and environmental conditions. However, our current understanding of physiological and pathological GSH changes at high spatial and temporal resolution is limited due to non-availability of practicable GSH-detection methods. In order to measure GSH at real-time, a ratiometric genetically encoded nanosensor was developed using fluorescent proteins and fluorescence resonance energy transfer (FRET) approach. The construction of the sensor involved the introduction of GSH binding protein (YliB) as a sensory domain between cyan fluorescent protein (CFP; FRET donor) and yellow fluorescent protein (YFP; FRET acceptor). The developed sensor, named as FLIP-G (Fluorescence Indicator Protein for Glutathione) was able to measure the GSH level under in vitro and in vivo conditions. When the purified FLIP-G was titrated with different concentrations of GSH, the FRET ratio increased with increase in GSH-concentration. The sensor was found to be specific for GSH and also stable to changes in pH. Moreover, in live bacterial cells, the constructed sensor enabled the real-time quantification of cytosolic GSH that is controlled by the oxidative stress level. When expressed in yeast cells, FRET ratio increased with the external supply of GSH to living cells. Therefore, as a valuable tool, the developed FLIP-G can monitor GSH level in living cells and also help in gaining new insights into GSH metabolism.
Mohammad Ahmad; Naser A. Anjum; Ambreen Asif; Altaf Ahmad. Real-time monitoring of glutathione in living cells using genetically encoded FRET-based ratiometric nanosensor. Scientific Reports 2020, 10, 1 -9.
AMA StyleMohammad Ahmad, Naser A. Anjum, Ambreen Asif, Altaf Ahmad. Real-time monitoring of glutathione in living cells using genetically encoded FRET-based ratiometric nanosensor. Scientific Reports. 2020; 10 (1):1-9.
Chicago/Turabian StyleMohammad Ahmad; Naser A. Anjum; Ambreen Asif; Altaf Ahmad. 2020. "Real-time monitoring of glutathione in living cells using genetically encoded FRET-based ratiometric nanosensor." Scientific Reports 10, no. 1: 1-9.
The generation of reactive oxygen species (ROS) (and their reaction products) in abiotic stressed plants can be simultaneous. Hence, it is very difficult to establish individual roles of ROS (and their reaction products) in plants particularly under abiotic stress conditions. It is highly imperative to detect ROS (and their reaction products) and ascertain their role in vivo and also to point their optimal level in order to unveil exact relation of ROS (and their reaction products) with the major components of ROS-controlling systems. Förster Resonance Energy Transfer (FRET) technology enables us with high potential for monitoring and quantification of ROS and redox variations, avoiding some of the obstacles presented by small-molecule fluorescent dyes. This paper aims to: (i) introduce ROS and overview ROS-chemistry and ROS-accrued major damages to major biomolecules; (ii) highlight invasive and non-invasive approaches for the detection of ROS (and their reaction products); (iii) appraise literature available on genetically encoded ROS (and their reaction products)-sensors based on FRET technology, and (iv) enlighten so far unexplored aspects in the current context. The studies integrating the outcomes of the FRET-based ROS-detection approaches with OMICS sciences (genetics, genomics, proteomics, and metabolomics) would enlighten major insights into real-time ROS and redox dynamics, and their signaling at cellular and subcellular levels in living cells.
Naser A. Anjum; Amreen; Aadil Y. Tantray; Nafees A. Khan; Altaf Ahmad. Reactive oxygen species detection-approaches in plants: Insights into genetically encoded FRET-based sensors. Journal of Biotechnology 2020, 308, 108 -117.
AMA StyleNaser A. Anjum, Amreen, Aadil Y. Tantray, Nafees A. Khan, Altaf Ahmad. Reactive oxygen species detection-approaches in plants: Insights into genetically encoded FRET-based sensors. Journal of Biotechnology. 2020; 308 ():108-117.
Chicago/Turabian StyleNaser A. Anjum; Amreen; Aadil Y. Tantray; Nafees A. Khan; Altaf Ahmad. 2020. "Reactive oxygen species detection-approaches in plants: Insights into genetically encoded FRET-based sensors." Journal of Biotechnology 308, no. : 108-117.
Nanotechnology is an emerging tool in the field of life sciences and has since continually proven its potential in contributing to sustainable competitiveness and growth in several industrial sectors. Though we have only managed to scrape the surface of the potential impact of nanotechnology in the coming years, the noteworthy findings, however, cannot be neglected. Recent years have seen a revolutionizing improvement in industrial sectors which has stemmed from the development of various nanosensors in an overwhelming proportion employing their ability to detect chemical or biological species on a nanoscale. A detailed study of nanosensors and its various types, namely, magnetic, chemical, mechanical, optical, thermal, and nanobiosensors, and their fabrication and application in the field of biotechnology has been discussed in the chapter allowing the reader to gain a better understanding of the new striking advancements in the field of research.
Ghazala Ambrin; Habiba Kausar; Ruphi Naz; Altaf Ahmad. Current Status of Nanosensors in Biological Sciences. Nanobiosensors for Agricultural, Medical and Environmental Applications 2020, 15 -41.
AMA StyleGhazala Ambrin, Habiba Kausar, Ruphi Naz, Altaf Ahmad. Current Status of Nanosensors in Biological Sciences. Nanobiosensors for Agricultural, Medical and Environmental Applications. 2020; ():15-41.
Chicago/Turabian StyleGhazala Ambrin; Habiba Kausar; Ruphi Naz; Altaf Ahmad. 2020. "Current Status of Nanosensors in Biological Sciences." Nanobiosensors for Agricultural, Medical and Environmental Applications , no. : 15-41.
Isoleucine is one of the branched chain amino acids that plays a major role in the energy metabolism of human beings and animals. However, detailed investigation of specific receptors for isoleucine has not been carried out because of the non-availability of a tool that can monitor the metabolic flux of this amino acid in live cells. This study presents a novel genetically-encoded nanosensor for real-time monitoring of isoleucine in living cells. This nanosensor was developed by sandwiching a periplasmic binding protein (LivJ) of E. coli between a fluorescent protein pair, ECFP (Enhanced Cyan Fluorescent Protein), and Venus. The sensor, named GEII (Genetically Encoded Isoleucine Indicator), was pH stable, isoleucine-specific, and had a binding affinity (Kd) of 63 ± 6 μM. The GEII successfully performed real-time monitoring of isoleucine in bacterial and yeast cells, thereby, establishing its bio-compatibility in monitoring isoleucine in living cells. As a further enhancement, in silico random mutagenesis was carried out to identify a set of viable mutations, which were subsequently experimentally verified to create a library of affinity mutants with a significantly expanded operating range (96 nM-1493 μM). In addition to its applicability in understanding the underlying functions of receptors of isoleucine in metabolic regulation, the GEII can also be used for metabolic engineering of bacteria for enhanced production of isoleucine in animal feed industries.
Shruti Singh; Maheshwar Prasad Sharma; Abdulaziz A Alqarawi; Abeer Hashem; Elsayed Fathi Abd Allah; Altaf Ahmad. Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor. Sensors 2019, 20, 146 .
AMA StyleShruti Singh, Maheshwar Prasad Sharma, Abdulaziz A Alqarawi, Abeer Hashem, Elsayed Fathi Abd Allah, Altaf Ahmad. Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor. Sensors. 2019; 20 (1):146.
Chicago/Turabian StyleShruti Singh; Maheshwar Prasad Sharma; Abdulaziz A Alqarawi; Abeer Hashem; Elsayed Fathi Abd Allah; Altaf Ahmad. 2019. "Real-Time Optical Detection of Isoleucine in Living Cells through a Genetically-Encoded Nanosensor." Sensors 20, no. 1: 146.
Cysteine plays a critical role in maintaining normal human metabolism, redox homeostasis, and immune regulation. Despite its functional versatility, cysteine metabolism in the human body is not well understood because of the lack of a robust tool for real-time measurement of cysteine at the cellular and sub-cellular level. In the present study, a genetically encoded nanosensor was developed using Cj0982 protein of Campylobacter jejuni, Enhanced Cyan Fluorescent Protein (ECFP) and Venus. The Cj0982 was sandwiched between ECFP and Venus for the construction of the nanosensor, named as Cys-FS (Cysteine-Fluorescent-Sensor). The Cys-FS is pH stable, specific to cysteine and has an affinity of 1.2 × 10−5 M. A range of affinity mutants were also developed with a cumulative cysteine detection range from 800 nM to 3.5 mM. The Cys-FS nanosensor was expressed in bacterial, yeast and mammalian cells, and the dynamics of cysteine level was measured in living cells using the confocal microscopy. The results showed that the Cys-FS nanosensor successfully monitored the dynamics of cysteine in both prokaryotic and eukaryotic systems without disrupting the cell. Thus, this study presents a novel nanosensor that can measure cysteine in living cells. This nanosensor is minimally invasive and non-toxic.
Shruti Singh; M.P. Sharma; Altaf Ahmad. Construction and characterization of protein-based cysteine nanosensor for the real time measurement of cysteine level in living cells. International Journal of Biological Macromolecules 2019, 143, 273 -284.
AMA StyleShruti Singh, M.P. Sharma, Altaf Ahmad. Construction and characterization of protein-based cysteine nanosensor for the real time measurement of cysteine level in living cells. International Journal of Biological Macromolecules. 2019; 143 ():273-284.
Chicago/Turabian StyleShruti Singh; M.P. Sharma; Altaf Ahmad. 2019. "Construction and characterization of protein-based cysteine nanosensor for the real time measurement of cysteine level in living cells." International Journal of Biological Macromolecules 143, no. : 273-284.
Nitrogen (N) is the basis of plant growth and development and, is considered as one of the priming agents to elevate a range of stresses. Plants use solar radiations through photosynthesis, which amasses the assimilatory components of crop yield to meet the global demand for food. Nitrogen is the main regulator in the allocation of photosynthetic apparatus which changes of the photosynthesis (Pn) and quantum yield (Fv/Fm) of the plant. In the present study, dynamics of the photosynthetic establishment, N-dependent relation with chlorophyll fluorescence attributes and Rubisco efficacy was evaluated in low-N tolerant (cv. CR Dhan 311) and low-N sensitive (cv. Rasi) rice cultivars under low-N and optimum-N conditions. There was a decrease in the stored leaf N under low-N condition, resulting in the decreased Pn and Fv/Fm efficiency of the plants through depletion in the activity and content of Rubisco. The Pn and Fv/Fm followed the parallel trend of leaf N content during low-N condition along with depletion of intercellular CO2 concentration and overall conductance under low-N condition. Photosynthetic saturation curve cleared abrupt decrease of effective quantum yield in the low-N sensitive rice cultivar than the low-N tolerant rice. Also, the rapid light curve highlighted the unacclimated regulation of photochemical and non-photochemical quenching in the low-N condition. The low-N sensitive rice cultivar triumphed non-photochemical quenching, whereas the low-N tolerant rice cultivar rose gradually during the light curve. Our study suggested that the quantum yield is the key limitation for photosynthesis in low-N condition. Regulation of Rubisco, photochemical and non-photochemical quenching may help plants to grow under low-N level.
Aadil Yousuf Tantray; Sheikh Shanawaz Bashir; Altaf Ahmad. Low nitrogen stress regulates chlorophyll fluorescence in coordination with photosynthesis and Rubisco efficiency of rice. Physiology and Molecular Biology of Plants 2019, 26, 83 -94.
AMA StyleAadil Yousuf Tantray, Sheikh Shanawaz Bashir, Altaf Ahmad. Low nitrogen stress regulates chlorophyll fluorescence in coordination with photosynthesis and Rubisco efficiency of rice. Physiology and Molecular Biology of Plants. 2019; 26 (1):83-94.
Chicago/Turabian StyleAadil Yousuf Tantray; Sheikh Shanawaz Bashir; Altaf Ahmad. 2019. "Low nitrogen stress regulates chlorophyll fluorescence in coordination with photosynthesis and Rubisco efficiency of rice." Physiology and Molecular Biology of Plants 26, no. 1: 83-94.
Ajmalicine is naturally present in the root bark of Catharanthus roseus L. and Rauvolfia serpentina (L.) Benth ex.Kurz. It has been extensively utilized in the treatment of hypertension across the world. The increased demand, overconsumption, and low content of the alkaloid in the plants have raised the issue of the depletion of natural sources. The metabolic engineering approach has not been successful in improving the content of the ajmalicine because the metabolic regulation of this metabolite is not known. The regulation of a metabolite in the metabolic pathway requires a tool that can carry out real-time measurement of the flux of the metabolite in living system. Given this, the present study was conducted to develop a genetically encoded FRET-based nanosensor by engineering human Cytochrome P450-2D6, an ajmalicine binding protein. The Cytochrome P450-2D6 was sandwiched between two FRET fluorophores. The design of the nanosensor brings two fluorescent proteins in conjunction with the ajmalicine binding protein, such that it undergoes FRET (Fluorescence Resonance Energy Transfer) upon binding of the ligand. The nanosensor, named as FLIP-Ajn (Fluorescence Indicator Protein for Ajmalicine), was pH stable and ajmalicine specific. The affinity of the FLIP-Ajn was 582 μM. The FLIP-Ajn successfully performed real-time measurement of ajmalicine in prokaryotic (bacteria) and eukaryotic systems (yeast, animal cell line, and plant suspension culture), thereby, establishing its biocompatibility in monitoring of ajmalicine in living cells. Besides, several affinity mutants of the nanosensor were generated through mutations in the ajmalicine binding protein to increase the detection range of the nanosensor at varying physiological scales. The non-invasiveness and high spatial and temporal resolution of the tool holds a great significance in the bio-imaging of a highly compartmentalized metabolic pathway. The flux study of ajmalicine will help in identifying the regulatory steps involved in the synthesis of the alkaloids and, hence, will improve the production rate of ajmalicine from its natural sources.
Ghazala Ambrin; Mohammad Ahmad; Abdulaziz A Alqarawi; Abeer Hashem; Elsayed Fathi Abd Allah; Altaf Ahmad. Conversion of Cytochrome P450 2D6 of Human Into a FRET-Based Tool for Real-Time Monitoring of Ajmalicine in Living Cells. Frontiers in Bioengineering and Biotechnology 2019, 7, 1 .
AMA StyleGhazala Ambrin, Mohammad Ahmad, Abdulaziz A Alqarawi, Abeer Hashem, Elsayed Fathi Abd Allah, Altaf Ahmad. Conversion of Cytochrome P450 2D6 of Human Into a FRET-Based Tool for Real-Time Monitoring of Ajmalicine in Living Cells. Frontiers in Bioengineering and Biotechnology. 2019; 7 ():1.
Chicago/Turabian StyleGhazala Ambrin; Mohammad Ahmad; Abdulaziz A Alqarawi; Abeer Hashem; Elsayed Fathi Abd Allah; Altaf Ahmad. 2019. "Conversion of Cytochrome P450 2D6 of Human Into a FRET-Based Tool for Real-Time Monitoring of Ajmalicine in Living Cells." Frontiers in Bioengineering and Biotechnology 7, no. : 1.
One of the major abiotic stresses that affect productivity of rice is salinity. Rice cultivars showed significant genetic variation in response to salt stress. In the present investigation, differential growth pattern and physio-chemical traits-based screening of high yielding rice cultivars of various salt affected areas of India was carried out, and salt-sensitive and salt-tolerant cultivars were identified. Differential responses of antioxidant enzyme activity and tolerance index at maximum level of salt treatment depicted that Jhelum and Vytilla-4 cultivars of rice were sensitive and tolerant to salt stress, respectively. Analysis of growth, morpho-physiological, and biochemical parameters also confirmed the salt-tolerant and salt-sensitive characters of cv. Vytilla-4 and cv. Jhelum, respectively. Nano-LCMS/MS-based proteome profile of these two cultivars was carried out to find out the mechanism lying behind the salt tolerance. A total number of 514 and 770 protein spots were reported in the most salt-tolerant (cv. Vytilla-4) and salt-sensitive (cv. Jhelum) cultivars, respectively. The differentially expressed proteins (DEPs) were found associated with major metabolic pathways including photosynthesis, energy metabolism, amino acid metabolism, nitrogen assimilation and stress and signalling pathways. The changes in the major proteins like Ribulose bisphosphate carboxylase small chain, chlorophyll a-b binding protein, phosphoglycerate kinase, cytochrome c oxidase subunit 5C, glutamine synthetase, glutathione S-transferase, peroxidase, and thioredoxin elucidated the mechanism activated by salt-tolerant cv. Vytilla-4. The transcriptional validation of some of the differentially expressed proteins through real-time quantitative PCR analysis further validated the proteomic results. Outcomes of this work could help in finding out the potential cross-links of different pathways involved in salt-tolerance mechanisms operating in the studied here rice cultivars under salt stress.
Arajmand Frukh; Tariq Omar Siddiqi; M. Iqbal R. Khan; Altaf Ahmad. Modulation in growth, biochemical attributes and proteome profile of rice cultivars under salt stress. Plant Physiology and Biochemistry 2019, 146, 55 -70.
AMA StyleArajmand Frukh, Tariq Omar Siddiqi, M. Iqbal R. Khan, Altaf Ahmad. Modulation in growth, biochemical attributes and proteome profile of rice cultivars under salt stress. Plant Physiology and Biochemistry. 2019; 146 ():55-70.
Chicago/Turabian StyleArajmand Frukh; Tariq Omar Siddiqi; M. Iqbal R. Khan; Altaf Ahmad. 2019. "Modulation in growth, biochemical attributes and proteome profile of rice cultivars under salt stress." Plant Physiology and Biochemistry 146, no. : 55-70.