Assessment of microwave drying for rapid and convenient analysis of medicinal plants for quality assurance.

INTRODUCTION: The safety and quality of many medicinally important herbs are compromised since farmers and small organizations are involved in the cultivation, aggregation, and primary processing of these herbs. Such organizations often lack adequate quality control facilities. To improve the safety and quality of herbal products, simple, rapid, and affordable quality control systems are required. OBJECTIVES: The aim of this study was to assess the suitability of microwave oven-drying for moisture content (MC) determination and sample preparation of herbs in small organizations. METHODS: Microwave oven-drying (720 W) and convective oven-drying at 105°C for MC determination were compared. The effects of three different drying methods (microwave oven-drying, low-temperature convective drying, and freeze-drying) on in vitro antioxidant and polyphenol oxidase (PPO) activity were determined, similarity analysis was conducted using HPLC signature spectra, and validation was performed with LC-MS focusing on one herb. RESULTS: Microwave oven-drying at 720 W significantly reduced the drying time (from hours to minutes), whereas the spatial variation of temperature in convective ovens set at 105°C can cause about 10% underestimation of MC. Microwave oven-drying showed similar macro-properties like freeze-drying and higher extractability (10%-20%) and in vitro antioxidant capacity (33%-66%) and lower PPO activity compared to low-temperature convective drying. HPLC signature spectra revealed strong similarity of soluble components between freeze-dried and microwave oven-dried herbs. LC-MS analysis demonstrated more common compounds between freeze-dried and microwave oven-dried Centella asiatica extracts, whereas convective tray-dried samples had fewer compounds common with samples obtained by freeze-drying or microwave oven-drying. CONCLUSIONS: Microwave oven-drying is rapid (tens of min) and shows small batch-to-batch variation compared to oven-drying at 105°C. The in vitro antioxidant assays and signature spectra can be used for assessing the source and purity or quality of a specific herb variety.

Truncated α-amylase: an improved candidate for textile processing.

Enzymes are indispensable biocatalysts required in various steps of textile processing to minimize various chemical-induced hazards. The present work focuses on the applications of the truncated α-amylase in textile industry for desizing of fabrics by starch hydrolysis. The multiple sequence alignment was performed to find homology and the possible truncation region in Bacillus subtilis MTCC 121 α-amylase with same bacilli family α-amylase. Two constructs were generated for α-amylase gene of Bacillus subtilis MTCC 121 (Amy_F, full-length and Amy_T, C-terminal truncated) were cloned, overexpressed, purified, and characterized. Results revealed that activity of Amy_T was found to be 2.87-fold better than Amy_F. Further, the optimum temperature of Amy_F and Amy_T was obtained at 45 °C and 55 °C, respectively, whereas optimum pH was recorded at pH 7 and pH 8, respectively. Improved thermostability of Amy_T was further confirmed through thermal shift assay. Subsequently, starch-coated fabrics were tested for starch removal using the α-amylases. Comparative analysis revealed that Amy_T performed better in starch removal from polystyrene (85%), silk (75%), and cotton (70%) fabrics. The removal of starch from the fabrics was further confirmed by FESEM. Conclusively, this work presents one truncated α-amylase as an improved candidate over its full-length counterpart for textile desizing.

Multifactorial level of extremostability of proteins: can they be exploited for protein engineering?

Research on extremostable proteins has seen immense growth in the past decade owing to their industrial importance. Basic research of attributes related to extreme-stability requires further exploration. Modern mechanistic approaches to engineer such proteins in vitro will have more impact in industrial biotechnology economy. Developing a priori knowledge about the mechanism behind extreme-stability will nurture better understanding of pathways leading to protein molecular evolution and folding. This review is a vivid compilation about all classes of extremostable proteins and the attributes that lead to myriad of adaptations divulged after an extensive study of 6495 articles belonging to extremostable proteins. Along with detailing on the rationale behind extreme-stability of proteins, emphasis has been put on modern approaches that have been utilized to render proteins extremostable by protein engineering. It was understood that each protein shows different approaches to extreme-stability governed by minute differences in their biophysical properties and the milieu in which they exist. Any general rule has not yet been drawn regarding adaptive mechanisms in extreme environments. This review was further instrumental to understand the drawback of the available 14 stabilizing mutation prediction algorithms. Thus, this review lays the foundation to further explore the biophysical pleiotropy of extreme-stable proteins to deduce a global prediction model for predicting the effect of mutations on protein stability.

An insight into plant lipase research - challenges encountered.

Lipases from bacterial, fungal, and animal sources have been purified to homogeneity with very few of them being contributed from plants. Plant lipases are mostly found in energy reserve tissues, for example, oilseeds. They act as biocatalysts which are attractive due to their high substrate specificity, low production cost and easy pharmacological acceptance due to their eukaryotic origin. Hence plant lipases represent better potential for commercial applications in organic synthesis, food, detergent and pharmacological industries. However, low expression, uneconomical fold purity and the plethora of difficulties related to their recombinant expression has been limiting their commercial applicability and posing challenges to many researchers. This article focuses on comprehensive approaches that have been reported to date to address these challenges.

Innovations and challenges in adsorption-based wastewater remediation: A comprehensive review.

Water contamination is an escalating emergency confronting communities worldwide. While traditional adsorbents have laid the groundwork for effective water purification, their selectivity, capacity, and sustainability limitations have driven the search for more advanced solutions. Despite many technological advancements, economic, environmental, and regulatory hurdles challenge the practical application of advanced adsorption techniques in large-scale water treatment. Integrating nanotechnology, advanced material fabrication techniques, and data-driven design enabled by artificial intelligence (AI) and machine learning (ML) have led to a new generation of optimized, high-performance adsorbents. These advanced materials leverage properties like high surface area, tailored pore structures, and functionalized surfaces to capture diverse water contaminants efficiently. With a focus on sustainability and effectiveness, this review highlights the transformative potential of these advanced materials in setting new benchmarks for water purification technologies. This article delivers an in-depth exploration of the current landscape and future directions of adsorbent technology for water remediation, advocating for a multidisciplinary approach to overcome existing barriers in large-scale water treatment applications.

Antibiotic persistence and its impact on the environment.

From boon molecules to molecules contributing to rising concern has been the sojourn of antibiotics. The problem of antibiotic contamination has gotten worse due to antibiotics' pervasive use in every aspect of the environment. One such consequence of pollution is the increase in infections with antibiotic resistance. All known antimicrobials being used for human benefit lead to their repetitive and routine release into the environment. The misuse of antibiotics has aggravated the situation to a level that we are short of antibiotics to treat infections as organisms have developed resistance against them. Overconsumption is not just limited to human health care, but also occurs in other areas such as aquaculture, livestock, and veterinary applications for the purpose of improving feed and meat products. Due to their harmful effects on non-target species, the trace level of antibiotics in the aquatic ecosystem presents a significant problem. Since the introduction of antibiotics into the environment is more than their removal, they have been given the status of persistent pollutants. The buildup of antibiotics in the environment threatens aquatic life and may lead to bacterial strains developing resistance. As newer organisms are becoming resistant, there exists a shortage of antibiotics to treat infections. This has presented a very critical problem for the health-care community. Another rising concern is that the development of newer drug molecules as antibiotics is minimal. This review article critically explains the cause and nature of the pollution and the effects of this emerging trend. Also, in the latter sections, why we need newer antibiotics is questioned and discussed.

Multitargeting: An Alternative Approach to Tackle Multidrug Resistance in Tuberculosis.

BACKGROUND: The prevalence of drug-resistant organisms has steadily increased over the past few decades worldwide. Especially in tuberculosis (TB) disease, the problems of co-morbidity and the rapid emergence of multidrug resistance have necessitated the development of multitarget-based therapeutic regimens. Several multitargeting compounds against Mycobacterium tuberculosis (Mtb) have been studied through novel in silico tools but these have rendered reduced efficacy in clinical trials. The authors have focussed on many exotic targets belonging to crucial Mtb survival pathways whose molecular structures and functions are underexplored. Likewise, insights into the hidden possibilities of promiscuous compounds from natural products or repurposed drugs to inhibit other cellular proteins apart from their validated targets are also depicted in this review. In addition to the existing line of drugs currently recommended for multidrug-resistant TB, newer host-directed therapies could also be fruitful. Furthermore, several challenges, including safety/efficacy ratios of multitarget compounds highlighted here, can also be circumnavigated by researchers to design "smart drugs" for improved tuberculosis therapeutics. CONCLUSION: A holistic approach towards alleviating the existing drawbacks of drug discovery in drug-resistant TB has been outlined. Finally, considering the current needs, the authors have put forward an overall summary of possible trends in multitargeting that are significant for futuristic therapeutic solutions.

Evaluation of selected antitumor agents as subversive substrate and potential inhibitor of trypanothione reductase: an alternative approach for chemotherapy of Leishmaniasis.

Trypanothione reductase (TryR) is a validated drug target against Leishmaniasis. Using integrated computational and experimental approaches, the authors report doxorubicin and mitomycin C, known antitumor agents, as novel inhibitors of TryR of leishmania parasite. Interestingly, these compounds also act as subversive substrates and subvert the physiological function of enzyme by converting it from an anti-oxidant to a pro-oxidant. Possible mechanism of subversive substrate is discussed. Both doxorubicin and mitomycin C show significant effect on redox homeostasis of the parasite and high-leishmanicidal activity. The toxicity studies as well as available toxicity data in literature indicate these compounds to have acceptable toxicity in limited dose.

In silico characterization of thermostable lipases.

Thermostable lipases are of high priority for industrial applications as they are endowed with the capability of carrying out diversified reactions at elevated temperatures. Extremophiles are their potential source. Sequence and structure annotation of thermostable lipases can elucidate evolution of lipases from their mesophilic counterparts with enhanced thermostability hence better industrial potential. Sequence analysis highlighted the conserved residues in bacterial and fungal thermostable lipases. Higher frequency of AXXXA motif and poly Ala residues in lid domain of thermostable Bacillus lipases were distinguishing characteristics. Comparison of amino acid composition among thermostable and mesostable lipases brought into light the role of neutral, charged and aromatic amino acid residues in enhancement of thermostability. Structural annotation of thermostable lipases with that of mesostable lipases revealed some striking features which are increment of gamma turns in thermostable lipases; being first time reported in our paper, longer beta strands, lesser beta-branched residues in helices, increase in charged-neutral hydrogen bonding pair, hydrophobic-hydrophobic contact and differences in the N-cap and C-cap residues of the α helices. Conclusively, it can be stated that subtle changes in the arrangement of amino acid residues in the tertiary structure of lipases contributes to enhanced thermostability.

In silico characterization of thermoactive, alkaline and detergent-stable lipase from a Staphylococcus aureus strain.

Bacterial true lipases having thermo and alkaline stability are highly attractive for their industrial production of pharmaceuticals, agrochemicals, cosmetics, and flavour. Staphylococcus aureus lipase (SAL3) remains active at temperatures 40-60°C, with an optimum temperature of 55°C and an optimum pH of 9.5 stable over a range of 5-12. Detailed understanding of the structure and insight into the activity of such lipase would aid in engineering lipases that would function in the desired extreme industrial environments. In the present study, we carried out in silico characterization and structural modeling of SAL3 which is thermoactive, alkaline and detergent-stable. Comparison of SAL3 with other staphylococcal lipases indicates that SAL3 is a true lipase having the catalytic triad (residues Ser119, Asp310 & His352) and the calcium binding site (residues Asp351, Asp354, Asp359, Asp362 and Gly286). Conservation in sequence implies that interfacial activation mechanism is possible in SAL3 with the lid formed by helix (residues 180-196) and loop (residues 197-206). Three dimensional (3D) structure model of SAL3 has been predicted for the first time and aims at understanding its function and biochemical characteristics of possessing relatively high thermal and pH stability.

Scaffold-based Screening and Molecular Dynamics Simulation Study to Identify Two Structurally Related Phenolic Compounds as Potent MMP1 Inhibitors.

BACKGROUND: Matrix metalloproteinase 1 are zinc-dependent endopeptidases responsible for the controlled breakdown of the extracellular matrix resulting in the maintenance of homeostasis. Dysregulation of MMP1 leads to the progression of various pathological conditions like cancer, rheumatoid arthritis, cardiovascular disease, skin damage and fibrotic disorder. Thus, MMP1 inhibition is the potential drug target of many synthetic MMP1 inhibitors but lack of substrate specificity hinders their clinical applicability. Hence, inhibitors from natural products have gained widespread attention. OBJECTIVE: The present study attempts screening of novel MMP1 inhibitors from the ZINC database based on experimentally reported natural inhibitors of MMP1 as a scaffold. METHODS: Molecular docking study was performed with 19 experimentally reported natural inhibitors spanning across nine different classes followed by virtual screening using the selected compounds. The selected compounds were subjected to molecular dynamics simulation. RESULTS: Twenty compounds were screened with a cut-off of -9.0 kcal/mol of predicted free energy of binding, which further converged to 6 hits after docking studies. After comparing the docking result of 6 screened hits, two best compounds were selected. ZINC02436922 had the best interaction with six hydrogen bond formation to a relatively confined region in the S1'site of MMP1 and -10.01 kcal/mol of predicted free energy of binding. ZINC03075557 was the secondbest compound with -9.57 kcal/mol predicted binding free energy. Molecular dynamics simulation of ZINC02436922 and ZINC03075557 corroborates docking study. CONCLUSION: This study indicated phenolic compounds ZINC02436922 and ZINC03075557 as potential MMP1 inhibitors.

A strategic approach of enzyme engineering by attribute ranking and enzyme immobilization on zinc oxide nanoparticles to attain thermostability in mesophilic Bacillus subtilis lipase for detergent formulation.

The present study envisaged rationalized protein engineering approach to attain thermostability in a mesophilic Bacillus subtilis lipase. Contributing amino acids for thermostability were analyzed from homologous thermophilic-mesophilic protein dataset through relative abundance and generated ranking model. Analyses divulged priority of charged amino acids for thermostability. Ranking model was used to predict thermostabilizing mutations. Three lipase mutants, bsl_the1 (V149K, Q150E), bsl_the2 (F41K, W42E, V149K, Q150E) and bsl_the3 (F41K, W42E, P119E, Q121K, V149K, Q150E) were generated and validated through in silico and in vitro approaches for improved activity and thermostability. ZnO nanoparticles were synthesized by precipitation method and functionalized using polyethylenimine, APTES and glutaraldehyde for lipase immobilization. The immobilization was confirmed through various analytical techniques. Analysis revealed bsl_wt showed optimum activity at 35 °C and pH 8 which was increased to 60 °C and pH 10 in case of ZnO-bsl_the3. The ZnO-bsl_the3 showed 80% of their initial activity after 60 days of storage stability and retained 78% of activity after 20 cycles of reuse. Lipases were applied for oil and grease stain removal from fabric. ZnO-bsl_the3 removed 90% and 82% of oil and grease stains, respectively. Conclusively, it revealed a promising perspective of low-cost nanobiocatalysts in detergent formulation.

RankProt: A multi criteria-ranking platform to attain protein thermostabilizing mutations and its in vitro applications - Attribute based prediction method on the principles of Analytical Hierarchical Process.

Attaining recombinant thermostable proteins is still a challenge for protein engineering. The complexity is the length of time and enormous efforts required to achieve the desired results. Present work proposes a novel and economic strategy of attaining protein thermostability by predicting site-specific mutations at the shortest possible time. The success of the approach can be attributed to Analytical Hierarchical Process and the outcome was a rationalized thermostable mutation(s) prediction tool- RankProt. Briefly the method involved ranking of 17 biophysical protein features as class predictors, derived from 127 pairs of thermostable and mesostable proteins. Among the 17 predictors, ionic interactions and main-chain to main-chain hydrogen bonds were the highest ranked features with eigen value of 0.091. The success of the tool was judged by multi-fold in silico validation tests and it achieved the prediction accuracy of 91% with AUC 0.927. Further, in vitro validation was carried out by predicting thermostabilizing mutations for mesostable Bacillus subtilis lipase and performing the predicted mutations by multi-site directed mutagenesis. The rationalized method was successful to render the lipase thermostable with optimum temperature stability and Tm increase by 20°C and 7°C respectively. Conclusively it can be said that it was the minimum number of mutations in comparison to the number of mutations incorporated to render Bacillus subtilis lipase thermostable, by directed evolution techniques. The present work shows that protein stabilizing mutations can be rationally designed by balancing the biophysical pleiotropy of proteins, in accordance to the selection pressure.

Alleviating the Neglected Tropical Diseases: Recent Developments in Diagnostics and Detection.

BACKGROUND: Neglected tropical diseases (NTDs) are communicable diseases caused by a group of bacteria, viruses, protozoa and helminths prevalent in more than 145 countries that affect the world's poverty stricken populations. WHO enlists 18 NTDs amongst people living in endemic areas having inaccessibility to preventive measures. Steps to reduce the global disease burden of the NTDs need attention at multi-factorial levels. Control programmes, mass drug administrations, transmission checks, eradication surveillances and diagnoses are some of them. The foremost in this list is confirmatory diagnosis. A comprehensive summary of the innovative, high-impact, multiplexed, low-cost diagnostic tools developed in the last decade that helped to meet the needs of users can depict a holistic approach to further evaluate potential technologies and reagents currently in research. Major Advancements: A literature survey based on developing nano-biotechnological platforms to meet the diagnostic challenges in NTDs towards development of a useful point-of-care (POC) unit is reported. However, in order to pave the way for complete eradication more sensitive tools are required that are user-friendly and applicable for use in endemic and low-resource settings. There are various novel research progresses/advancements made for qualitative and quantitative measurement of infectious load in some diseases like dengue, Chagas disease and leishmaniasis; though further improvements on the specificity and sensitivity front are still awaited. Strategies to combat the problem of antimicrobial drug resistance in diagnosis of NTDs have also been put forward by various research groups and organizations. Moreover, the state-of-the-art "omics" approaches like metabolomics and metagenomics have also started to contribute constructively towards diagnosis and prevention of the NTDs. CONCLUSION: A concrete solution towards a single specimen based common biomarker detection platform for NTDs is lacking. Identifying robust biomarkers and implementing them on simple diagnostic tools to ease the process of pathogen detection can help us understand the obstacles in current diagnostic measures of the NTDs.

Multisubstrate specific flavin containing monooxygenase from Chlorella pyrenoidosa with potential application for phenolic wastewater remediation and biosensor application.

Microbial degradation of phenolic pollutants in industrial wastewater is dependent on enzymatic pathway comprising a cascade of phenol metabolizing enzymes. Phenol hydroxylase is the first enzyme of the pathway catalysing the initial attack on phenol in green algae Chlorella pyrenoidosa. The present work reports cost-effective production of partially purified microalgal phenol hydroylase by single-step purification and characterization of its kinetic properties with the view of application for enzyme-based remediation of phenolic wastewater or in phenolic biosensor. The enzyme with a molecular weight of 25 kDa shows all characteristics of phenol hydroxylases, that is, hydroxylation of phenol to catechol (confirmed by HPLC), substrate-dependent NADPH oxidation, absorption spectrum typical of flavoproteins and peptide mass fingerprint corresponding to flavoprotein hydroxylase. The enzyme utilizes phenol with apparent Michealis constant (Km) of 1.71 µM, maximal velocity (Vmax) of 0.4 µM/min with optimal activity at pH 7 and 35°C. Fe2+chelators (Phenanthroline and sodium arsenate), heavy metals, denaturants and oxidizing agents showed inhibitory effect on phenol hydroxylation activity of the enzyme. The enzyme has broad substrate specificity against isomeric diphenols, isomeric methylphenols, halogen-substituted phenols, amino-substituted phenols, nitrophenols, hydroxybenzaldehyde and hydroxylbenzoic acid. The enzyme shows remarkable storage stability at room temperature and at 4°C. The multisubstrate specificity coupled to remarkable storage stability of the microalgal phenol hydroxylase opens up avenues for its application in remediation of a wide range of phenolics released in industrial wastewater or phenolic biosensor application.

Deciphering the rationale behind specific codon usage pattern in extremophiles.

Protein stability is affected at different hierarchies - gene, RNA, amino acid sequence and structure. Gene is the first level which contributes via varying codon compositions. Codon selectivity of an organism differs with normal and extremophilic milieu. The present work attempts at detailing the codon usage pattern of six extremophilic classes and their harmony. Homologous gene datasets of thermophile-mesophile, psychrophile-mesophile, thermophile-psychrophile, acidophile-alkaliphile, halophile-nonhalophile and barophile-nonbarophile were analysed for filtering statistically significant attributes. Relative abundance analysis, 1-9 scale ranking, nucleotide compositions, attribute weighting and machine learning algorithms were employed to arrive at findings. AGG in thermophiles and barophiles, CAA in mesophiles and psychrophiles, TGG in acidophiles, GAG in alkaliphiles and GAC in halophiles had highest preference. Preference of GC-rich and G/C-ending codons were observed in halophiles and barophiles whereas, a decreasing trend was reflected in psychrophiles and alkaliphiles. GC-rich codons were found to decrease and G/C-ending codons increased in thermophiles whereas, acidophiles showed equal contents of GC-rich and G/C-ending codons. Codon usage patterns exhibited harmony among different extremophiles and has been detailed. However, the codon attribute preferences and their selectivity of extremophiles varied in comparison to non-extremophiles. The finding can be instrumental in codon optimization application for heterologous expression of extremophilic proteins.

Identification of Xanthine Derivatives as Inhibitors of Phosphodiesterase 9A Through In silico and Biological Studies.

BACKGROUND: In recent times, computer aided methodologies have received broad attention in drug development. These studies have improved the accuracy and shortened the time frame to identify suitable drug candidates from large datasets. Xanthine is a plant alkaloid which also acts as an intermediate product on the pathway of purine degradation. Xanthine acts as scaffold for various natural and synthetically derived bioactive molecules. OBJECTIVE: The present work aims to screen xanthine derivatives targeting phosphodiesterase 9A (PDE9A), one of the most important regulatory protein of signal transduction. METHOD: In silico approach such as Virtual screening, molecular docking and molecular dynamic was attempted to screen a repertoire of 2055 xanthine derivatives extracted from ZINC database against PDE9A. The potency of the resultant screened compound was finally validated by spectrophotometric malachite green inhibition assay. RESULTS: Preliminary virtual screening narrowed down the compounds to a list of 10 which is followed by a second round of stringent screening using molecular docking approach. Top four hits were selected for thorough interaction analysis with PDE9A. The molecular docking analysis of best ranked compound, ZINC62579975 (-12.59) revealed its potential to establish essential chemical interactions with inhibition determining key residues in the PDE9A active site. The stability of ZINC62579975 in PDE9A was further validated by 6 ns molecular dynamic simulation studies. The in vitro malachite spectrophotometric assay confirmed the bioactive potential of the above compound. Comparative inhibition studies asserted more potency of ZINC62579975 towards PDE9A (46.96 ± 1.78 µM) than PDE5A (61.023 ± 1.71 µM) and PDE4D (70.04 ± 1.98 µM). CONCLUSION: The entire study validates ZINC62579975 as a potent candidate molecule for PDE9A inhibition. The present study provides a roadmap for future drug designing of more potent xanthine derivatives. This study also explores the potential of xanthine scaffold in future drug development process.

Xanthine scaffold: scope and potential in drug development.

Medicinal plants have been the basis for discovery of various important marketed drugs. Xanthine is one such lead molecule. Xanthines in various forms (caffeine, theophylline, theobromine, etc) are abode in tea, coffee, cocoa, chocolate etc. giving them popular recognition. These compounds are best known for their diverse pharmaceutical applications as cyclic nucleotide phosphodiesterase inhibition, antagonization of adenosine receptor, anti-inflammatory, anti-microbial, anti-oxidant and anti-tumor activities. These properties incentivize to use xanthine as scaffold to develop new derivatives. Chemical synthesis contributes greater diversity in xanthine based derivatisation. With highlighting the existing challenges in chemical synthesis, the present review focuses the probable solution to fill existing lacuna. The review summarizes the available knowledge of xanthine based drugs development along with exploring new xanthine led chemical synthesis path for bringing diversification in xanthine based research. The main objective of this review is to explore the immense potential of xanthine as scaffold in drug development.

The Metabolomic Strategy in Tuberculosis Therapy.

Tuberculosis causes around 1.4 million deaths every year remaining to be a major cause of morbidity and mortality. Mycobacterium tuberculosis is adept at surviving the assault of our immune system, which has perplexed many researchers. Not only are they difficult to treat, but also difficult to detect accurately. The current reservoir of drugs and method to diagnose them is depleting faster than we are able to replace them with newer one. The advances made in the field of metabolomic have opened up a window of opportunities such as identification of new drug and druggable site, prediction of drug toxicity and detection of new biomarkers to accurately diagnose the disease. With its short history of less than two decades, metabolomics has shown very promising future to tackle this concern. Equally important developments in analytical techniques and application of data interpretation have further facilitated the scope of this field. The present review discusses the advances in metabolomic research with an emphasis on tuberculosis diagnosis and therapeutics.

Hairpin stabilized fluorescent silver nanoclusters for quantitative detection of NAD+ and monitoring NAD+/NADH based enzymatic reactions.

A set of 90 mer long ssDNA candidates, with different degrees of cytosine (C-levels) (% and clusters) was analyzed for their function as suitable Ag-nanocluster (AgNC) nucleation scaffolds. The sequence (P4) with highest C-level (42.2%) emerged as the only candidate supporting the nucleation process as evident from its intense fluorescence peak at λ660 nm. Shorter DNA subsets derived from P4 with only stable hairpin structures could support the AgNC formation. The secondary hairpin structures were confirmed by PAGE, and CD studies. The number of base pairs in the stem region also contributes to the stability of the hairpins. A shorter 29 mer sequence (Sub 3) (ΔG = -1.3 kcal/mol) with 3-bp in the stem of a 7-mer loop conferred highly stable AgNC. NAD+ strongly quenched the fluorescence of Sub 3-AgNC in a concentration dependent manner. Time resolved photoluminescence studies revealed the quenching involves a combined static and dynamic interaction where the binding constant and number of binding sites for NAD+ were 0.201 L mol-1 and 3.6, respectively. A dynamic NAD+ detection range of 50-500 µM with a limit of detection of 22.3 µM was discerned. The NAD+ mediated quenching of AgNC was not interfered by NADH, NADP+, monovalent and divalent ions, or serum samples. The method was also used to follow alcohol dehydrogenase and lactate dehydrogenase catalyzed physiological reactions in a turn-on and turn-off assay, respectively. The proposed method with ssDNA-AgNC could therefore be extended to monitor other NAD+/NADH based enzyme catalyzed reactions in a turn-on/turn-off approach.